JP2024500921A - Novel isomeric compounds containing ring-opened thiosuccinimide groups, oligopeptide fragments and chiral moieties - Google Patents

Abstract

TECHNICAL FIELD The present disclosure relates to the field of medicinal chemistry, and in particular to methods for separating isomeric compounds containing open ring thiosuccinimide groups and chiral moieties. [Selection diagram] Figure 48

Description

The present disclosure relates to the field of medicinal chemistry, and in particular to isomeric compounds with open ring thiosuccinimide groups, oligopeptide fragments and chiral moieties that have anticancer activity. The invention also relates to a method for separating isomeric compounds.

Increased tumor recurrence and severe side effects of chemotherapy drugs in mammals have reduced the clinical efficacy of many currently used anticancer drugs. Therefore, there is always a need to develop alternative or synergistic anti-cancer drugs that minimize side effects. One of the key strategies for developing effective anti-cancer drugs is the study of anti-cancer drugs derived from drug "combinations", and hundreds of trials are currently being conducted to evaluate novel combinations of targeted drugs. has been done. Such targeted agents include antibody-drug-conjugates.

The concept of antibody-drug conjugates (ADCs) is not new and has been proposed as a potent antitumor drug based on monoclonal antibodies, combining antibodies (as targeting and/or therapeutic moieties) with conventional cytotoxic drugs (with strong cytotoxicity). It combines the advantages of

An ideal ADC linker must be sufficiently stable outside the cell to ensure that the small molecule drug is linked to the ligand, and once in the cell, the cleavable linker must be able to be used under appropriate conditions. the requirement that the active ingredient be cleaved below to release the active small molecule drug and that, in the case of non-cleavable linkers, the active ingredient is typically a small molecule, the linker and the ligand consist of amino acid residues resulting from enzymatic hydrolysis. need to be met. Therefore, in the development of ADCs, linker design and associated coupling strategies are of great importance, and they not only play an important role in stabilizing ADCs, but also affect the biological activity of the conjugate, the aggregation state, In vivo bioavailability, distribution and metabolism are also directly affected.

Current mainstream coupling technologies are chemical coupling strategies mainly based on lysine or cysteine residues in antibodies. Due to the diversity in the number and position of amino acids in the antibody that can react with the linker, the number and position of cytotoxins in the ADC is variable and the ADC thus obtained is heterogeneous. Such heterogeneity can affect the quality, stability, efficacy, metabolism and toxicity of ADCs; for example, the drug description for ADC Kadcyla, launched in 2013, states that the number of cytotoxins per antibody is It is specified that the number is from 0 to 8, with an average n of about 3.5. The problem of heterogeneity in ADCs is a major challenge in the development of new generation ADCs.

One of the deficiencies of bioconjugates is off-target release, which results in toxicity to normal tissues and reduces the number of effective bioconjugates at the target, reducing efficacy. More than half of the antibody-drug conjugates (ADCs) on the market or in clinical trials use thiosuccinimide structures (thiosuccinimide linkages) to conjugate small molecule drugs to target antibodies or proteins. However, the thiosuccinimide linkage is not stable and in vivo, reverse Michael addition or exchange with other thiol groups can occur, directly leading to shedding of the cytotoxin from the ADC and causing off-target toxicity.

Ring opening of the succinimide can increase the stability of the bioconjugate by removing potential sites for thiol group exchange via reverse Michael addition or reverse Michael addition mechanisms.

Thiosuccinimide structures are formed by the reaction of thiol groups and maleimide structures. In situations where regioselectivity is low or incomplete, the ring-opening reaction yields a pair of regioisomers. Additionally, a chiral center is created by the covalent bond between the thiol group and succinimide. The presence of diastereomers and enantiomers poses major challenges in the work-up of ring-opening reactions. Therefore, further research on isomers is needed and new purification methods are urgently needed.

式中、
ペイロード、Ｌ^１、Ｌ^２及びＭは以下で定義する。 The present disclosure relates to a compound having a structure of any one of the following formulas (XI) to (XIV).

During the ceremony,
The payloads, L ¹ , L ² and M are defined below.

The present disclosure further provides antibody drug conjugates (ADCs) prepared using a compound of formula (XI), (XII), (XIII), or (XIV) and an antibody or antigen-binding fragment thereof.

式中、ｘは－ＯＨ又は－ＮＨ_２である。 In one particular embodiment, the compounds of formulas (XI) to (XIV) have structures of formulas (i) to (iv), respectively;

In the formula, x is -OH or _-NH2 .

The mixture of ADCs obtained by conjugating a compound of formula (iii) or a compound of formula (iv) with a therapeutic antibody is designated as "α-ADC".

α-ADCs containing ring-opened thiosuccinimide groups, oligopeptide fragments, and chiral moieties have been prepared, and their biological activity has been demonstrated by in vitro and in vivo assays. The unexpected anti-cancer results are as follows.

α-ADC has a significant inhibitory effect on the proliferation of HER2-positive cells, and the effect is several tens of times that of DM1.

α-ADC can be concentrated in tumors within 24 hours and maintain stability in situ.

In summary, under the current study conditions, repeated intravenous infusions (once every 3 weeks, 3 times in total) with 10, 30, and 45 mg/kg α-ADC were tolerated by cynomolgus monkeys, and a single dose It can be 45 mg/kg. The highest non-severely toxic dose (HNSTD) was determined to be 45 mg/kg, whereas Kadcyla exhibits a toxic dose of 10 mg/kg.

The optimized molecular structure improved metabolism. Compared to Kadcyla, α-ADC showed similar effects at lower doses.

The present disclosure further provides a method of separating one or more target compounds from a mixture 1, wherein the mixture 1 includes four compounds, and each of the four compounds includes moiety 1, moiety 2, and moiety 3. .

ただし、
部分１中のチオール基とアミド基は２つの連結部位を構成し、部分２はその一方を介して部分１に連結され、部分３はその他方を介して部分１に連結され、
部分２は１つ又は複数のキラル中心を含み、
前記４つの化合物中の部分１は異なり、
部分３は前記分子中の残りの部分であり、
部分３の分子量は１９００以下であり、
前記１つ又は複数の標的化合物は混合物１に含まれる４つの化合物から選択さる。 Portion 1 has an open ring thiosuccinimide structure selected from formulas (I) to (IV).

however,
The thiol group and the amide group in moiety 1 constitute two linking sites, moiety 2 is linked to moiety 1 through one of them, moiety 3 is linked to moiety 1 through the other,
Portion 2 contains one or more chiral centers;
Moiety 1 in the four compounds is different,
Part 3 is the remaining part in the molecule,
The molecular weight of portion 3 is less than or equal to 1900;
The one or more target compounds are selected from the four compounds contained in mixture 1.

The method includes:
(1) preparing mixture 1;
(2) chromatography of mixture 1 to obtain the target compound;
however,
a. each of said target compounds is obtained as a separate product, or b. The two target compounds are obtained as a mixture, which is defined as mixture 2.

In one embodiment, the method further includes steps (3) and (4) below.
(3) collecting an eluate containing mixture 3 collected in step (2), where mixture 3 contains one or more additional target compounds different from the target compounds separated in step (2); include.
(4) The eluate collected in step (3) is subjected to chromatography to separate the additional target compound.

Depending on the position of the thiol group, formulas (I) and (II) are referred to as β, and the configurations of formulas (III) and (IV) are referred to as α.

The present disclosure further provides a method for analyzing one or more compounds in mixture 1, wherein mixture 1 includes four compounds, and each of the four compounds in said mixture is part 1, part 2, and Contains part 3.

The present disclosure further provides a method for analyzing one or more compounds in mixture 2, said mixture comprising two compounds, each of the two compounds in said mixture being part 1, part 2 and Contains part 3.

FIG. 3 is an HPLC diagram of the separation of β1, β2 and α isomers using Daisopak C4 SP-120-8-C4-P. FIG. 3 is an HPLC diagram of the separation of β1, β2 and α isomers using Kromasil C18 100-10C18. FIG. 3 is an HPLC diagram of the separation of β1, β2 and α isomers using Acchrom C18 C18-ME 10um 100A. FIG. 3 is an HPLC diagram of the separation of β1, β2 and α isomers using Daisopak C18 100-10-ODS-P. Figure 3 is an HPLC diagram of the separation of β1, β2 and α isomers, where eluent A is 0.1% TFA in water and eluent B is MeOH. HPLC diagram of the separation of β1, β2 and α isomers, where eluent A is 0.1% aqueous H ₃ PO ₄ and eluent B is MeOH. HPLC diagram of the separation of β1, β2 and α isomers, where eluent A is 0.2% TFA in water and eluent B is MeOH. HPLC diagram of the separation of β1, β2 and α isomers, where eluent A is 0.1% aqueous CH ₂ O ₂ and eluent B is MeOH. HPLC diagram of separation of β1, β2 and α isomers where eluent A is 0.2% CH ₂ O ₂ and eluent B is MeOH. Figure 3 is an HPLC diagram of the separation of β1, β2 and α isomers, where eluent A is 0.5% AA aqueous solution and eluent B is MeOH. Figure 3 is an HPLC diagram of the separation of β1, β2 and α isomers, where eluent A is 0.1% aqueous CH ₃ COOH and eluent B is MeOH. [0034] FIG. 3 is an HPLC diagram of the separation of β1, β2 and α isomers, where eluent A is 0.3% aqueous CH ₃ COOH and eluent B is MeOH. HPLC diagram of the separation of β1, β2 and α isomers where eluent A is 0.3% CH ₃ COOH + 0.05% L-TA in water and eluent B is MeOH. HPLC diagram of the separation of β1, β2 and α isomers where eluent A is 10mM NaH ₂ PO ₄ PH=2.0 and eluent B is MeOH. HPLC diagram of the separation of β1, β2 and α isomers where eluent A is 10mM NaH ₂ PO ₄ PH=4.0 and eluent B is MeOH. HPLC diagram of the separation of β1, β2 and α isomers where eluent A is 10mM NaH ₂ PO ₄ PH=6.0 and eluent B is MeOH. HPLC diagram of the separation of β1, β2 and α isomers where eluent A is 10 mM K ₂ HPO ₄ PH=4.0 and eluent B is MeOH. HPLC diagram of separation of β1, β2 and α isomers where eluent A is 10mM TEAP PH=4.0 and eluent B is MeOH. HPLC diagram of the separation of β1, β2 and α isomers where eluent A is 10 mM NaClO ₄ PH=2.0 and eluent B is MeOH. HPLC diagram of separation of β1, β2 and α isomers where eluent A is 0.3% aqueous CH ₃ COOH and eluent B is MeOH with gradient from 46% to 66% B in 30 min. . HPLC diagram of separation of β1, β2 and α isomers where eluent A is 0.3% aqueous CH ₃ COOH and eluent B is MeOH with gradient from 46% to 56% B in 80 min. . FIG. 3 is an HPLC diagram of the analysis of β1, β2 and α isomers using Phenomenex Gemini 110 Å 5 um 250*4.6 mm. α1 and α2 using Kromasil 100-10C18, 21.2*250mm (100Å, 10μm) where eluent C is 0.1% TFA aqueous solution and eluent D is CAN containing 0.1% TFA. FIG. 3 is an HPLC diagram of the separation of isomers. HPLC diagram of separation of α1 and α2 isomers, where eluent C is 0.1% TFA aqueous solution and eluent D is ACN. HPLC diagram of separation of α1 and α2 isomers, where eluent C is 0.1% AcOH aqueous solution and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 0.3% AcOH aqueous solution and eluent D is ACN. FIG. 3 is an HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 0.1% aqueous CH ₂ O ₂ and eluent D is ACN. FIG. 3 is an HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 0.3% aqueous CH ₂ O ₂ and eluent D is ACN. HPLC diagram of separation of α1 and α2 isomers, where eluent C is 0.5% AA aqueous solution and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 0.1% H ₃ PO ₄ aqueous solution and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 0.2% H ₃ PO ₄ aqueous solution and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 10 mM NaH ₂ PO ₄ PH=2.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 10 mM NaH ₂ PO ₄ PH=4.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 10 mM NaH ₂ PO ₄ PH=6.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 10 mM K ₂ HPO ₄ PH=2.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 10 mM K ₂ HPO ₄ PH=4.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 10 mM K ₂ HPO ₄ PH=6.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 10 mM K ₂ HPO ₄ PH=8.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 30 mM NaH ₂ PO ₄ PH=2.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 30 mM NaH ₂ PO ₄ PH=6.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 30 mM NaH ₂ PO ₄ PH=4.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 10 mM TEAP PH=6.0 and eluent D is ACN. HPLC diagram of the separation of α1 and α2 isomers, where eluent C is 10 mM TEAP PH=2.0 and eluent D is ACN. HPLC diagram of separation of α1 and α2 isomers where eluent C is 10mM TEAP PH=4.0 and eluent D is ACN. FIG. 2 is an HPLC diagram for analysis of α1 and α2 isomers using Waters X-select, CSH Phenyl-Hexyl 3.5um 150*4.6mm. Figure 3 shows the effect of isomers on cancer cell proliferation. The effect of α-ADC on cancer cell proliferation is shown. The biodistribution properties of α-ADC (PET/CT scan results after a single intravenous injection of ⁸⁹ Zr-α-ADC) are shown. The in vivo antitumor effect of α-ADC is shown, the x-axis represents time after a single administration, and the y-axis represents tumor size. The pharmacokinetic properties of α-ADC are shown, with the x-axis representing time after a single intravenous injection and the y-axis representing serum concentration. A summary of the toxicokinetic data of α-ADC is shown, where the x-axis represents the time after the first dose, 0 h, 504 h and 1008 h represent the time points of repeated administration of intravenous infusion, 1008 h The 2016th hour represents a 6-week recovery period. The y-axis represents serum concentration.

In Figures 1-45, the x-axis represents time (minutes). In Figures 1-21 and 45, the y-axis represents UV absorbance (mAU), and in Figures 22-44, the y-axis represents UV absorbance (AU).

General Definitions Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. As used herein, technology refers to technology as commonly understood in the art and includes variations and equivalent substitutions that are obvious to those skilled in the art. Although the following terms will be readily understood by those skilled in the art, the following definitions are provided to better explain this disclosure. When a trade name is mentioned herein, it is intended to refer to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated by reference in their entirety.

The fact that a particular amount, concentration, or other value or parameter is indicated as a range, preferred range, or preferred upper limit or preferred lower limit does not mean that any upper limit or preferred value and any lower limit or preferred value and should be understood as equivalent to what is specifically disclosed, whether or not explicitly stated. Unless otherwise stated, numerical ranges recited herein are intended to include the endpoints of the range and all whole numbers and fractions within the range. For example, the expression "about 0.01% to about 1%" means any value between 0.01% and 1%, such as 0.01%, 0.05%, 0.1%, 0. 15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0. 65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95% and 1%. Other similar expressions such as "40%-50% to 50%-70%" should be understood in the same way.

Unless expressly stated otherwise herein, singular forms such as "a" and "such" include plural references. The expression “one or more” or “at least one” means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. The above (species) can be expressed.

When used in conjunction with numerical variables, the terms "about" and "approximately" generally mean that the value of the variable and all values of the variable are within experimental error (e.g., within a 95% confidence interval of the mean) or This means within ±10% of the designated value or within a wider range.

The term "mixture" refers to a mixture that includes one or more compounds, one or more of which can be a target compound. The term "target compound" refers to a compound to be separated or purified. When defining a separation process, target compounds are determined before the separation operation. It should be understood that the product containing the target compound can be in any desired form, such as a product containing pure isomeric compounds or a mixture containing a plurality of predetermined target compounds.

The term "stoichiometric ratio" refers to the ratio of certain weights of various substances.

The term "any" or "at any" means that the subsequently described event may occur, but does not necessarily occur; This includes cases where this happens or cases where it does not occur.

The expression "comprising" or similar expressions "comprising," "containing," "having," and the like are open-ended and do not exclude additional unlisted elements, steps, or ingredients. The expression "consisting of" excludes any element, step or ingredient not explicitly indicated. The expression "consisting essentially of" extends the scope to the specified element, step or component, and any present element, step or ingredient that does not substantially affect the essential features and novel features of the claimed subject matter. It means to be limited to the ingredients. The expression "comprising" should be understood to include the expressions "consisting essentially of" and "consisting of."

で表すことができる。実線で描かれた不斉原子への結合は、該原子上の全ての可能な立体異性体（例えば、特定のエナンチオマー、ラセミ混合物等）が考慮されていることを示すことを意図する。波線で描かれた不斉原子への結合は、該結合が

であることを示すことを意図する。ソリッドウェッジ又は破線ウェッジで表される不斉原子への結合は、示されている立体異性体の存在を示すことを意図する。ラセミ混合物に存在する場合、ソリッドウェッジ又は破線ウェッジは、絶対立体化学ではなく相対立体化学を定義するために用いられる。別段の説明がない限り、本開示の化合物は、立体異性体（シス及びトランス異性体、光学異性体（例えば、Ｒ及びＳ鏡像異性体）、ジアステレオマー、幾何異性体、回転異性体、配座異性体、アトロプ異性体及びそれらの混合物を含む）の形態で存在し得る。本開示の化合物は、上記異性体形態の１つ又は複数を示すことができ、それらの混合物（例えば、ラセミ混合物及び／又はジアステレオマー対）からなることができる。 The chemical bonds in the compounds of this disclosure are defined herein as

It can be expressed as A bond to an asymmetric atom drawn as a solid line is intended to indicate that all possible stereoisomers on that atom (eg, specific enantiomers, racemic mixtures, etc.) are considered. A bond to an asymmetric atom drawn with a wavy line indicates that the bond is

is intended to indicate that Bonds to asymmetric atoms represented by solid wedges or dashed wedges are intended to indicate the presence of the indicated stereoisomer. When present in a racemic mixture, a solid or dashed wedge is used to define relative stereochemistry rather than absolute stereochemistry. Unless otherwise specified, the compounds of this disclosure include stereoisomers (cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotamers, conformational isomers). (including loci isomers, atropisomers and mixtures thereof). The compounds of the present disclosure may exhibit one or more of the above-mentioned isomeric forms and may consist of mixtures thereof (eg, racemic mixtures and/or diastereomeric pairs).

The term "hydrocarbyl group" refers to a monovalent group derived from a hydrocarbon. Examples of hydrocarbyl groups include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, and aryl groups.

The term "alkyl group" means a straight or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, the saturated aliphatic hydrocarbon group being connected to the rest of the molecule by a single bond. ing. Alkyl may contain 1 to 20 carbon atoms, ie may be C ₁ -C ₂₀ alkyl, for example a C ₁ -C ₄ alkyl group, a C ₁ -C ₃ alkyl group, a C ₁ -C ₂ alkyl group , C ₃ alkyl group, C ₄ alkyl group, and C ₃ -C ₆ alkyl group. Non-limiting examples of alkyl include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl. group, 1-methylbutyl group, 1-ethylpropyl group, 1,2-dimethylpropyl group, neopentyl group, 1,1-dimethylpropyl group, 4-methylpentyl group, 3-methylpentyl group, 2-methylpentyl group, 1-methylpentyl group, 2-ethylbutyl group, 1-ethylbutyl group, 3,3-dimethylbutyl group, 2,2-dimethylbutyl group, 1,1-dimethylbutyl group, 2,3-dimethylbutyl group, 1, Including, but not limited to, 3-dimethylbutyl group, or 1,2-dimethylbutyl group, or isomers thereof.

The term "valence state of a group" refers to the monovalent, divalent or trivalent nature of the group. A divalent group refers to a group obtained by removing one hydrogen atom from a carbon atom with free valence electrons from a corresponding monovalent group, and a trivalent group refers to a group obtained from a corresponding monovalent group by removing one hydrogen atom from a carbon atom with free valence electrons. Refers to a group obtained by removing one hydrogen atom from a carbon atom that has free valence electrons. For example, "alkylene group" refers to a straight or branched saturated divalent hydrocarbyl group. Examples of alkylene groups include methylene group (-CH ₂ -), ethylene group (-C ₂ H ₄ -), propylene group (-C ₃ H ₆ -), butylene group (-C ₄ H ₈ -), and pentylene group. group (-C ₅ H ₁₀ -), hexylene group (-C ₆ H ₁₂ -), 1-methylethylene group (-CH(CH ₃ )CH ₂ -), 2-methylethylene group (-CH ₂ CH(CH ₃ )-), methylpropylene group, and ethylpropylene group, but are not limited thereto. Examples of cycloalkylene groups include divalent monocyclic alkyl groups such as cyclopropylene group, cyclobutylene group, cyclopentylene group, cyclohexylene group, cycloheptylene group, and cyclooctylene group, and fused rings, Including, but not limited to, divalent polycyclic alkyl groups containing spiro rings or bridged rings. Examples of arylene groups include, but are not limited to, phenylene groups. Examples of heterocyclylene groups include, but are not limited to, pyrrolidinylene, imidazolidinylene, pyrazolidinylene, piperidinylene, piperazinylene, and morpholinyl. Examples of heteroarylene groups include pyrrolilene group, furanylene group, thienylene group, imidazolylene group, oxazorylene group, oxadiazolylene group, oxatriazolene group, isoxazolylene group, thiazolylene group, thiadiazolylene group, isothiazolylene group, and pyrazolylene group. including, but not limited to, triazolyl, triazolyl, and tetrazolylene groups.

である。 The term "opened thiosuccinimide group" refers to the product resulting from the opening of the succinimide ring in a thiosuccinimide group. The ring-opening reaction of the thiosuccinimide group can occur by cleavage of any one of the two amide bonds in the thiosuccinimide group, producing two isomers. In particular, the ring-opened thiosuccinimide group is

It is.

The term "regioselectivity" refers to a chemical reaction that can occur at multiple sites on a particular molecule, in which a reagent reacts preferentially to one atom over another, and the reaction occurs in preference to another regioisomer. It means that a certain positional isomer is preferentially produced. "Regiospecific" refers to the selectivity of a chemical reaction or reagent to target only one of two or more possible sites on a particular molecule.

The term "diastereomer" refers to stereoisomers whose molecules have two or more chiral centers and which are non-mirror images.

Small molecule compounds refer to molecules that are comparable in size to organic molecules commonly used in drugs. The term does not include biopolymers (eg proteins, nucleic acids, etc.), but includes low molecular weight peptides such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. or derivatives thereof. Usually, the molecular weight of the small molecule compound is, for example, about 100 to about 2000 Da, about 200 to about 1000 Da, about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, about 200 to about It can be 500 Da. As used herein, small molecule compounds are also referred to as drugs.

[0091] As used herein, the term "antibody (Ab)" refers to an immunoglobulin (Ig) molecule or derivative thereof that specifically binds to an antigen through at least one antigen binding site. . A "conventional" or "full length" antibody generally consists of four polypeptides: two heavy chains (HC) and two light chains (LC). As used herein, the definition of "antibody" includes conventional antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, diabodies or nanobodies (i.e., single domain antibodies, VHH domains); , but not limited to. Any member of an immunoglobulin type (e.g. IgG, IgM, IgD, IgE, IgA and IgY), any class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass (e.g. IgG2 and IgG2b), or Also contemplated are any derivatives of.

As used herein, an "antibody fragment" of an antibody refers to any portion of an antibody that contains fewer amino acid residues than a full-length antibody, e.g., at least a portion of a variable structural domain (e.g., an antigen-binding fragment that contains one or more CDRs and specifically binds to the same cognate antigen as the full-length antibody, or an Fc fragment that contains the heavy chain constant region of an antibody and binds to a cell surface Fc receptor. be. Antibody fragments can be obtained by various methods such as chemical or enzymatic treatment, chemical synthesis or recombinant DNA technology. Examples of antibody fragments include Fv (fragment variable region), scFv (single chain Fv fragment), dsFv (variable fragment stabilized by disulfide bonds), scdsFv (variable fragment stabilized by single chain disulfide bonds), including diabodies, Fd (hard fragments), Fab (antigen binding fragments), scFab (single chain Fabs), Fab', F(ab') ₂ , Fc (crystallizable region fragments) and any derivatives thereof. , but not limited to.

HER2 refers to human epidermal growth factor receptor 2, which belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family. In this application, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably.

As used herein, the term "natural amino acids" refers to amino acids that are constituent amino acids of proteins, including the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine , histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) and, less commonly, selenocysteine and pyrrolidine. As used herein, the term "unnatural amino acid" refers to an amino acid that is not a constituent amino acid of proteins. Specifically, the term refers to amino acids that are not naturally occurring amino acids as defined above.

When specifying amino acid sequences herein, the standard single letter amino acid code is used, unless otherwise indicated. Thus, for example, LPXTG represents the sequence -Leu-Pro-X-Thr-Gly-, where X is any natural or unnatural single amino acid residue, and LPETG represents the sequence -Leu-Pro-X-Thr-Gly- Gly-, LPETGG represents -Leu-Pro-Glu-Thr-Gly-Gly-, and GGG represents -Gly-Gly-Gly-.

As used herein, the term "peptide mimetic" refers to a compound that mimics the conformation and desired characteristics of a particular peptide.

As used herein, "receptor" refers to a structure present within or on a cell that binds to a specific substance and causes a specific effect within the cell. Receptors can include T cell receptors, B cell receptors, and receptors for signaling molecules, cell growth factors and cytokines, as described herein.

As described herein, interactions between chemicals (e.g. ions, molecules, groups or groups of atoms) refer to non-covalent interactions, including hydrogen bonds, π-π stacking, ionic interactions, ion-induced dipoles, etc. including, but not limited to, magnetic forces and ionic dipole forces.

As used herein, the term "ionization" refers to dissociating one or more acidic hydrogens in an acidic group to form one or more negatively charged groups, or one or more Refers to the bonding of multiple protons to a basic group to form one or more positively charged groups. For example, an "ionizable" carboxyl group can form a negatively charged group (-COO ^- ), and an "ionizable" amino group can form a positively charged group (-NH ₃ ⁺ ). can do. Molecules that simultaneously have one or more ionizable acidic groups and one or more ionizable basic groups can form zwitterions.

The term "reversed phase chromatography" (RPC) includes any chromatographic method that uses a hydrophobic stationary phase and a polar mobile phase, said hydrophobic stationary phase being more resistant to less hydrophobic or polar molecules. have affinity.

"Reversed Phase Liquid Chromatography" (RPLC) is a technique widely used in biopharmaceutical research and manufacturing. Stationary phases commonly used in RPLC are RP-modified silica gels, for example silica gels containing hydrocarbyl chains such as alkyl chains and/or cycloalkyl groups or aryl groups (e.g. phenyl, pentafluorophenyl, cyclohexyl groups). The group is grafted. A "monofunctional" alkyl silica gel stationary phase refers to a stationary phase obtained from the reaction of one molecule of silane with one silanol group on a silica gel support. A "bidentate" bonded alkyl silica gel stationary phase refers to an alkyl silica gel stationary phase obtained from the reaction of one molecule of bidentate silane containing two reactive groups with two silanol groups on a silica gel support. A bidentate stationary phase having two functional groups (eg, an alkyl group such as a methyl group, n-butyl group, n-octyl group or n-octadecyl group) on one bidentate silane is also called a difunctional stationary phase. Examples of alkyl silica gel stationary phases, such as alkyl-bonded silica gels, include, but are not limited to, silica gels that are bonded to C18, C8, C4 or C1 alkyl groups, particularly C18 alkyl groups. Alkyl bond The alkyl group in the silica gel may be linear or branched. In one embodiment, the alkyl group in the alkyl-bonded silica gel is a straight chain C18 alkyl group (n-octadecyl group). In one embodiment, the C18 alkyl groups in the alkyl-bonded silica gel are branched and have one or more side groups selected from C1-C6 alkyl groups (preferably isopropyl groups, isobutyl groups) and any combinations thereof. Contains chain alkyl groups. Some alkyl silica gel stationary phases contain embedded functional groups, such as carbamate groups (eg, Symmetry Shield ^™ RP C18), amide groups (eg, Zorbax Bonus-RP C18), and the like. Mixed mode modified silica gels are also used for RPLC, e.g. silica gels partially endcapped with hydrocarbyl groups and containing a proportion of residual (uncapped) silica silanols, and partially endcapped with hydrocarbyl groups. For example, NH ₂ -modified silica gel is end-capped and contains a fixed proportion of modified polar groups. The RP-modified silica gel can be particles of any suitable shape, given that robust chromatographic separations can be achieved with appropriate equipment. For example, alkyl-bonded silica gels can be generally spherical particles. In some cases, the RP-modified silica gel is surface porous or completely porous (surface and interior). In another embodiment, the RP-modified silica gel has a controlled surface porosity, with a pore size of 10 Å to 1 μm, preferably 20 to 600 Å, more preferably 40-300 Å, such as 60 Å, 70 Å, 80 Å, They are 90 Å, 100 Å, 110 Å, 120 Å, 130 Å, 140 Å, 150 Å, 160 Å, 170 Å, 180 Å, 190 Å, 200 Å, and 300 Å. In a further embodiment, the RP-modified silica gel has a controlled surface porosity and the pore size of the RP-modified silica gel is as described above.

The mobile phase for RPLC may be a mixed solvent of an organic solvent and water. In some literature, organic solvents are also referred to as "organic modifiers." Examples of aqueous binary mobile phases for RPLC include, but are not limited to, ACN/H ₂ O, MeOH/H ₂ O, i-PrOH/H ₂ O or THF/H ₂ O. Examples of aqueous ternary mobile phases for RPLC include, but are not limited to, THF/MeOH/ _H2O .

The mechanism of RPLC is generally related to the aspects of stationary phase and solute interactions controlled by changing the polarity of the mobile phase and solute-solvent and solvent-solvent dispersion interactions. Solute retention theory in liquid chromatography has been developed since the 1960s. Retention models based on molecular interactions, such as solute-solvent interactions (Scott-Kucera interaction model), solute-solvent interactions and localization of solute and/or solvent during adsorption (linear solvent strength (LSS) model) Efforts have been made to establish the Snyder-Soczewinski model (also known as the Snyder-Soczewinski model). However, given the many factors and parameter variability that influence chromatographic retention, the details of LC retention mechanisms are generally still unclear. During the development of "high pressure chromatography", the use of small particle column fillers and high column pressures improved both the precision and robustness of chromatography, e.g. solvatochromic analysis (linear solvation-energy relationship). Several new retention modeling methods have been reported, such as a method based on LSER, LSER). The RPLC mechanism for peptides is related to the size of the peptide, and the mechanism for oligopeptides is similar to other small molecules. Several semi-empirical models have been proposed to predict peptide retention, and several calculated "coefficients" are used to represent the interaction strength of the interaction between amino acid residues and the stationary phase. These coefficients do not distinguish between hydrophobic, hydrophilic, ionic and other interactions. However, even with these developments, there is still no predictive model that is satisfactory in real experimental settings. Successful prediction of chromatographic behavior for mass identification of unknown small or large molecules has not been reported.

"Hydrophobicity" is the association of non-polar groups or molecules in an aqueous environment resulting from the tendency of water to exclude non-polar molecules, and is understood as a measure of the relative tendency of solutes to prefer non-aqueous to aqueous environments; or a measure of the tendency of two (or more) solute molecules to aggregate in an aqueous solution. "Lipophilicity" refers to the affinity of a molecule or moiety for a lipophilic environment, typically in liquid-liquid (e.g. partition coefficient in 1-octanol/water) or solid-liquid (e.g. TLC or RP HPLC) systems. It is measured by the distribution behavior in a two-phase system that is a retention) system. The terms "lipophilic" and "hydrophobic" when used herein to describe a particular chemical entity represent the same characteristic of that chemical entity and therefore can be used interchangeably.

The retention factor (k) of a compositional component can be determined from the chromatogram using the formula k = (t _R - t _M )/t _M = (V _R - V _M )/V _M , where t _R and _VR are the retention time and retention volume of the component, respectively, _tM is the retention time of the component that is not retained, such as air or a solvent peak that is not retained or a marker that is not retained, and _VM is the retention time of the component that is not retained. This is the volume of mobile phase required to elute the constituent components. The logarithmic form of the retention factor, log k, is used as an index of lipophilicity. However, for quantifying lipophilicity, log k is not as important a parameter as log P. Although in some cases log k shows good correlation with shake flask distribution data (log P), clear differences are shown between slow equilibration and chromatographic distribution systems.

Chromatographic resolution (R) is calculated using the formula R = 2 (t _R2 - t _R1 )/(W ₁ + W ₂ ), where t _R2 and t _R1 are the retention of the two composition components. time, and W ₂ and W ₁ are the corresponding widths of the peak base obtained by extrapolating the relatively straight side of the peak to the baseline.

As used herein, the expression "sufficient chromatographic separation" and similar expressions refers to the chromatographic resolution (R) >0.8, preferably >0.9, e.g. >1.0, > 1.1, >1.2, >1.3, >1.4, >1.5, >1.6, >1.7, >1.8, >1.9 or >2.0 refers to something. In one particular embodiment, R>1.0. In another specific embodiment, R>1.5. Those skilled in the art can set and adjust the expected values of chromatography results according to different requirements and situations. It should be understood that in some cases the purification need not be complete (100% pure), but rather a predetermined purity determined prior to separation (e.g. >99%, >98%, >95%, >90%, >85%, >80%, >75%, >70%, >65% or >60%) can be considered sufficient.

In RPLC of ionizable solutes, the retention time of the solute is longest for the non-ionized form and shortest for the ionized form. When changing the pH of the mobile phase (eg, in an elution series), the retention time of the ionizable solute is always between the retention times of the ionized and non-ionized forms.

An elution gradient in RPLC generally refers to a gradient of increasing organic solvent content or changing pH value over time. The peak compression phenomenon is a characteristic of gradient separations. In the process of gradient separation, the movements of solutes at the front of the peak (close to the column outlet) and at the tail (close to the column inlet) are different. If the solute at the back of the peak moves faster than at the front, compression of the peak width is seen. Therefore, by adjusting the organic solvent content/mobile phase pH value, not only the retention time but also the peak width can be adjusted. However, it should be noted that changes in peak width due to peak compression phenomena in pH gradient separation cannot be predicted accurately.

The pH gradient of the mobile phase is the result of parameters including, for example, solute attributes, content of acidic/basic substances, temperature, and proportion of organic solvent when the mobile phase is partially aqueous.

The specific pKa values or ranges of pKa values described in this disclosure refer to values measured in aqueous solution. For pKa values not disclosed in this disclosure, reference may be made to those published by IUPAC, for example, in Ionization Constants of Organic Acids in Aqueous Solution, Sergeant, E.; P. , Dempsey B. , IUPAC Chemical Data Series No. 23, 1979. New York, New York: Pergamon Press, Inc. , p. 989 can be referred to. A particular pH value or pH value range refers to the apparent pH value when the liquid or solution referred to is only partially aqueous.

In industry, chromatography methods have been developed at different pressure ranges, such as, for example, normal pressure chromatography, medium pressure chromatography and high pressure chromatography. The term "overpressure chromatography" refers to a chromatography method that applies pressure greater than ambient pressure. "High pressure chromatography" refers to a chromatographic method in which a mobile phase and a liquid or suitably dissolved solid sample are passed through a column at high pressure, where "high pressure" refers to the flow of a mobile phase to particles of a stationary phase at a desired flow rate. , and the particles are typically smaller in size than those applied in atmospheric or medium pressure chromatography. In one embodiment, the stationary phase used in the RPLC method of the present disclosure is a RP-modified silica gel with a particle size of 1-300 μm, preferably 1-200 μm, more preferably 1-100 μm, even more preferably 1-300 μm. 80 μm, more preferably 3 to 60 μm, for example, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 20 μm, 40 μm, 50 μm, 60 μm.

Reversed-phase ion-pair (RPIP) chromatography (RPIPc), also known as ion-pair reversed-phase chromatography (IPRP), is a method in which a small amount of ion pairs is added to the mobile phase (e.g., by adding an ion-pair reagent). ) refers to a chromatographic method that increases the retention of strongly polar compounds. Retention of the resulting ion pairs is controlled by pH, counterion concentration and mobile phase polarity. In some cases, strongly polar compounds are charged molecules and are not retained by typical reversed phase chromatography. The mechanism of RPLC does not exclude the possible influence of ion pairs. The presence of acidic or basic substances in the RPLC mobile phase influences the chromatographic behavior of the solute with respect to the pH value of the environment, e.g. by forming ion pairs with the solute in ionized form. It is possible to give

The term "chromatographic process" is an abstract term and generally refers to a separation process using chromatographic methods. The separation process can include operations for separation and, optionally, operations for cleaning, equilibration or regeneration of the chromatography medium.

A "separation range" of eluent refers to the eluent used for separation, and an "equilibration range" of eluent refers to the eluent used for equilibration. It is to be understood that a separation range can represent a composition of the eluent that changes over time, and that said change can be terminated by the end of a time window, such as a critical event such as completion of elution of the target product. .

Methods of the Disclosure Methods of Separation In a first aspect, the present disclosure provides a method of separating one or more target compounds from a mixture 1, wherein the mixture 1 comprises four compounds, the four Each of the compounds includes moiety 1, moiety 2 and moiety 3.

ただし、
部分１中のチオール基とアミド基は２つの連結部位を構成し、部分２はその一方を介して部分１に連結され、部分３はその他方を介して部分１に連結され、
部分２は１つ又は複数のキラル中心を含み、
前記４つの化合物中の部分１は異なり、
部分３は前記分子中の残りの部分であり、
部分３の分子量は１９００以下であり、
前記１つ又は複数の標的化合物は混合物１に含まれる４つの化合物から選択される。 Portion 1 has an open ring thiosuccinimide structure selected from formulas (I) to (IV).

however,
The thiol group and the amide group in moiety 1 constitute two linking sites, moiety 2 is linked to moiety 1 through one of them, moiety 3 is linked to moiety 1 through the other,
Portion 2 contains one or more chiral centers;
Moiety 1 in the four compounds is different,
Part 3 is the remaining part in the molecule,
The molecular weight of portion 3 is less than or equal to 1900;
The one or more target compounds are selected from the four compounds contained in mixture 1.

The method includes:
(1) preparing mixture 1;
(2) chromatography of mixture 1 to obtain the target compound;
however,
a. each of said target compounds is obtained as a separate product, or b. The two target compounds are obtained as a mixture, which is defined as mixture 2.

Depending on the position of the thiol group, formulas (I) and (II) are referred to as β, and the configurations of formulas (III) and (IV) are referred to as α.

In one embodiment, the method further includes steps (3) and (4) below.
(3) collecting an eluate containing mixture 3 collected in step (2), where mixture 3 contains one or more additional target compounds different from the target compounds separated in step (2); include.
(4) The eluate collected in step (3) is subjected to chromatography to separate the additional target compound.

In one embodiment, step (1) is optional. In another embodiment, Mixture 1 is prepared by synthetic methods known in the art.

In one embodiment, each of the one or more target compounds is primarily in pure or substantially pure isomeric form, e.g. substantially free of other isomeric forms, e.g. more than 90% , such as having a purity of greater than 95%, such as greater than 98%, such as at least 99%.

1. Separation Step In one embodiment, moiety 1 in each target compound is different.

In one embodiment, in step (2) and step (4), four target compounds are obtained, two of which are obtained as separate products in step (2), and the other two target compounds are obtained as separate products in step (2). Obtained in step (4) as separate products.

In a preferred embodiment, moiety 1 in the two target compounds obtained as separate products in step (2) each has formula (I) or formula (II), and in step (4) the moieties 1 in the two target compounds are obtained as separate products. Moiety 1 in the two target compounds obtained as follows has formula (III) or formula (IV), respectively.

In another preferred embodiment, in step (2), the three target compounds contained in mixture 1 are obtained as separate products, and the remaining one is obtained in step (4).

In a further preferred embodiment, four target compounds are obtained in step (2), two of which are obtained as separate products and the remaining two target compounds are obtained as mixture 2. In a more preferred embodiment, moiety 1 in the two target compounds obtained as separate products in step (2) each has formula (I) or formula (II) and in the two obtained as mixture 2 Moiety 1 in the target compound has formula (III) or formula (IV), respectively.

2. Stationary Phase In one embodiment, the chromatography in step (2) and step (4) is reversed phase chromatography. In another embodiment, the stationary phases used in the reversed phase chromatography of steps (2) and (4) are each independently selected from alkyl-bonded silica gels.

The alkyl-bonded silica gel used in this disclosure is not particularly limited. Any alkyl silica gel stationary phase can be used provided that acceptable chromatographic results are obtained. In one preferred embodiment, the stationary phase used in the reverse phase chromatography of steps (2) and (4) is C18-bonded silica gel (ie, C18 alkyl-bonded silica gel). In one preferred embodiment, the alkyl group in the C18 bonded silica gel is a straight chain C18 alkyl (n-octadecyl group). In one embodiment, the C18-bonded silica gel is a generally spherical particle. In another embodiment, the C18-bonded silica gel has a controllable surface porosity, with a pore size of 20-600 Å, preferably 40-300 Å, more preferably 40-200 Å, such as 60 Å, 100 Å, 110 Å, 120 Å. , 150 Å, 200 Å, and 300 Å.

3. Mobile Phase In one embodiment, the mobile phase in step (2) is as follows.
As the composition of the eluent,
Eluent A is water optionally containing acidic agent 1;
Eluent B is an organic solvent 1 optionally containing an acidic agent 2;
However, the condition is that at least one of acidic agent 1 and acidic agent 2 is present,
For the separation range, B is a slope from about 0% to 30% to about 30% to 100%, and the rest is A;
The acidic agent 1 and the acidic agent 2 are independently inorganic acids or organic acids.

In one embodiment, organic solvent 1 is selected from methanol and ACN. In one preferred embodiment, organic solvent 1 is methanol.

In one embodiment, for the separation range of the mobile phase in step (2), B is a gradient of about 40%-50% to about 50%-70%, and the remainder is A.

In one embodiment, at least one of acidic agent 1 and acidic agent 2 is present. In one preferred embodiment, acidic agent 1 and acidic agent 2 are both present.

In one embodiment, the amounts of Acidic Agent 1 and Acidic Agent 2 (if present) are such that the gradient of Eluent C and Eluent D is accompanied by the gradient of Acidic Agent 1 and Acidic Agent 2.

In one embodiment, acidic agent 1 has the structure R ^c (COOH) _d , where d is 1 or 2, and R ^c is a C _1-15 hydrocarbyl group or a hydrocarbylene group; optionally substituted by at least one substituent selected from R ^y , R ^y is selected from -OH, -OCH ₃ , -OCH ₂ CH ₃ , -SH, -SCH ₃ , preferably -OH . In one preferred embodiment, the C _1-15 hydrocarbyl group in R ^c is a C _1-15 alkyl group, an alkylene group, a C _1-15 alkenyl group, or a C _1-15 alkenylene group, and is selected from R ^y optionally substituted with 1, 2, 3 or 4 substituents. In another preferred embodiment, d is 1 and the C _1-15 hydrocarbyl group in R ^c is a C _1-10 alkyl group, preferably a C _1-4 alkyl group. In one preferred embodiment, the C _1-15 hydrocarbyl group in R ^c is a C _1-2 alkyl group. In one particular embodiment, the C _1-15 hydrocarbyl group in R ^c is a methyl group. In one preferred embodiment, d is 2 and the C _1-15 hydrocarbyl group in R ^c is a C _2-4 alkylene group, especially an ethylene group, optionally substituted by one or two -OH groups. Ru.

In one embodiment, acidic agent 1 is selected from organic acids. In one embodiment, the pKa value of Acidic Agent 1 is 0.1-6.5, such as 0.4-6.5, 0.6-6.5, 1.0-6.5, 2.0 6.5, or 2.0 to 5.5. In a preferred embodiment, acidic agent 1 is selected from organic acids, and the pKa value of acidic agent 1 is between 4.0 and 5.5, such as about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5, especially about 4.7 or about 4.8, such as about 4.71, about 4.72, about 4.73, about 4.74, about 4.75, about 4.76, about 4.77, about 4.78, or about 4.79. In another preferred embodiment, acidic agent 1 is selected from organic acids, and acidic agent 1 has a pKa value of 2.3 to 3.8, such as about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, or about 3.8, especially about 3.0 or about 3.1, such as about 3.01, about 3.02, about 3.03, about 3.04, about 3.05, about 3.06, about 3.07, about 3.08, or about 3.09.

In another embodiment, acidic agent 1 is selected from AcOH and L-tartaric acid (L-TA). In one preferred embodiment, acidic agent 1 is AcOH. In one embodiment, the amount of Acidic Agent 1 is about 0.01% to about 1% based on the total volume of Eluent A. In one preferred embodiment, the amount of Acidic Agent 1 is from about 0.05% to about 0.5%, preferably from about 0.1% to about 0.5%, more preferably from about 0.1% to about 0.5%, based on the total volume of Eluent A. is about 0.1% to about 0.3%, especially 0.3%.

In one embodiment, acidic agent 2 is not present. In another embodiment, the type of acidic agent 2 is similar to acidic agent 1.

In one embodiment, the amount of acidic agent 2 is about 0.01% to about 1% based on the total volume of eluent B. In another embodiment, the amount of acidic agent 2 is from about 0.05% to about 0.5%, preferably from about 0.1% to about 0.5%, more preferably from about 0.1% to about 0.5%, based on the total volume of eluent B. is about 0.1% to about 0.3%, especially 0.3%.

In one embodiment, when eluting the target compound, the total amount of acidic agent 1 and acidic agent 2 is about 1% or less, preferably about 0.8% or less, based on the total volume of eluent A and eluent B, More preferably it is about 0.6% or less, for example about 0.4% or less, especially about 0.285% or less.

In one embodiment, the total content of Acidic Agent 1 and Acidic Agent 2 (if present) in the elution composition is about 0.005-0.06M.

In an alternative embodiment, the contents of Acidic Agent 1 and Acidic Agent 2 are adjusted to obtain a targeted pH profile, e.g. a targeted isocratic pH value or a targeted pH gradient. Dynamically configured and/or adjusted.

In one embodiment, the pH of the mobile phase is about 1.0 to about 4.0. In another embodiment, the pH of the mobile phase is about 1.0 to about 3.5. In one embodiment, the pH of the mobile phase is about 2.0 to about 3.5. In another embodiment, the pH of the mobile phase is about 2.4 to about 3.2.

In one embodiment, the mobile phase in step (4) is as follows.
As the composition of the eluent,
Eluent C is water optionally containing acidic agent 3;
Eluent D is an organic solvent 2 optionally containing an acidic agent 4,
For the separation range, D is a slope from about 0% to 30% to about 30% to 100%, and the rest is C;
The acidic agent 3 and the acidic agent 4 are independently inorganic acids or organic acids.

In one embodiment, organic solvent 2 is selected from methanol and ACN. In one preferred embodiment, organic solvent 2 is ACN.

In one embodiment, at least one of acidic agent 3 and acidic agent 4 is present. In one preferred embodiment, acidic agent 3 and acidic agent 4 are both present.

In one embodiment, for the separation range of the mobile phase in step (4), D is a gradient from about 10% to 30% to about 30% to 95%, and the remainder is C.

In one embodiment, the amounts of acidic agent 3 and acidic agent 4 (if present) are such that the gradient of eluent C and eluent D is accompanied by the gradient of acidic agent 3 and acidic agent 4.

The substances of the acidic agent 3 and the acidic agent 4 are not particularly limited as long as sufficient chromatographic separation can be achieved.

In one embodiment, acidic agent 2 is selected from organic acids. In another embodiment, the pKa value of Acidic Agent 2 is from 0.1 to 6.5, such as about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5 be.

In one embodiment, acidic agent 3 and acidic agent 4 are independently selected from TFA, phosphate buffer, ammonium acetate (AA), AcOH, H ₃ PO ₄ , TEAP, and L-tartaric acid (L-TA). Ru. In one preferred embodiment, the acidic agent 3 is selected from TFA, phosphate buffer, AA and TEAP. In a more preferred embodiment, the acidic agent 3 is selected from TFA, phosphate buffer and TEAP, especially TFA.

In one embodiment, acidic agent 4 is not present. In another embodiment, the type of acidic agent 4 is similar to acidic agent 3.

In one embodiment, the amount of acidic agent 3 is about 0.01% to about 1% based on the total volume of eluent C. In another embodiment, the amount of acidic agent 3 is from about 0.05% to about 0.5%, preferably from about 0.05% to about 0.3%, particularly from about 0.05% to about 0.3%, based on the total volume of eluent C. .1%.

In one embodiment, the amount of acidic agent 4 is about 0.01% to about 1% based on the total volume of eluent D. In another embodiment, the amount of acidic agent 4 is about 0.05% to about 0.5%, preferably about 0.05% to about 0.3%, based on the total volume of eluent D; In particular, it is 0.1%.

In one embodiment, for the separation range of the mobile phase in step (4), D is a gradient from about 10% to 30% to about 30% to 95%, and the remainder is C.

In one embodiment, the total content of acidic agent 3 and acidic agent 4 (if present) in the elution composition is about 0.01-0.25M.

In an alternative embodiment, the contents of acidic agent 3 and acidic agent 4 are adjusted dynamically to obtain a targeted pH profile, e.g. a targeted isocratic pH value or a targeted pH gradient. set and/or adjusted.

In one embodiment, the pH of the mobile phase is about 1.0 to about 6.0. In one specific embodiment, the pH of the mobile phase is about 2.0 to about 5.0, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0.

In one embodiment, isocratic pH values are applied, said isocratic pH values being in the range of about 2.0 to about 6.0. In another embodiment, the isocratic pH value of the mobile phase is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 5.0, or about 6.0. It is 0.

The method further comprises an optional equilibration step before step (2), wherein the mobile phase is as defined above, and the mobile phase equilibration range is about 100% to 95% A and about 0 % to 5% B, for example about 95% A and about 5% B. In another alternative embodiment, the method further comprises an optional equilibration step before step (4), the mobile phase is as defined above, and the mobile phase equilibration range is about 100% to 95% C and about 0% to 5% D, for example about 95% C and about 5% D.

4. Solute (target compound)
In one embodiment, the log P (octanol-water partition coefficient) value of Portion 2 is about 1 to about 5, preferably about 1.5 to about 4.5, about 2 to about 4.5, or about 2. 5 to about 4.5, for example, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2 , about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2 , about 4.3, about 4.4, or about 4.5.

式中、Ｑは、少なくとも１つのキラル中心を含む基であり、
Ｌ^１は存在しないか、又はＣ_１－１０アルキレン基、Ｃ_３－１０シクロアルキレン基、Ｃ_６－_１０アリーレン基、４～１０員ヘテロシクリレン基、５～１０員ヘテロアリーレン基、－ＮＨ－、－（ＣＯ）－、－ＮＨ（ＣＯ）－、及び－（ＣＯ）ＮＨ－から選択される１つ又は複数の二価基である。 In one embodiment, portion 2 has the following formula (V):

where Q is a group containing at least one chiral center,
L ¹ is absent or C _1-10 alkylene group, C _3-10 cycloalkylene group, C _6-10 arylene group, _4-10 membered heterocyclylene group, 5-10 membered heteroarylene group, -NH- , -(CO)-, -NH(CO)-, and -(CO)NH-.

In one embodiment, Q is _{a C5-50} hydrocarbyl group;
One or more "CH ₂ " structures in the hydrocarbyl group may be -O-, -S-, -NH-, -C(=O)-, -, provided that a stable structure is formed. S(=O)-, -S(=O) ₂ -, -NH(C=O)-, -C(=O)NH-, -NH-, -S(=O)-, -S(= O) NH-, -NHS(=O) ₂ -, or -S(=O) ₂ NH-,
one or more "CH" structures in said hydrocarbyl group are optionally replaced by N or P, provided that a stable structure is formed;
one or more carbon, sulfur, nitrogen or phosphorus atoms in Q are independently optionally substituted by oxo (=O),
Q is optionally substituted with at least one substituent selected from R ^q , each independently R ^a1 , ^{-OR a1} , -SR ^a1 , -NR ^a1 R ^b1 , -C(=O )OR ^a1 , -C(=O)NR ^a1 R ^b1 , -C(=O)R ^a1 , -S(=O) ₂ OR ^a1 , -S(=O) ₂ R ^a1 , -S(=O) ₂ NR ^a1 R ^b1 , -S(=O)R ^a1 , -C(=S)OR ^a1 , -C(=S)NR ^a1 R ^b1 , -C(=S)R ^a1 , -P(=O) (OR ^a1 )OR ^b1 , -C(=NR ^a1 )NR ^b1 R ^c1 ,
R ^a1 , R ^b1 and R ^c1 are each independently selected from hydrogen and a C _1-6 alkyl group.

In one embodiment, each cycloalkyl group structure in Q is optionally independently replaced by a heterocyclic structure having the same group valence state and the same number of ring atoms as said cycloalkyl group structure.

In one embodiment, each aryl group structure in Q is independently optionally replaced by a heteroaryl group structure having the same group valence state and the same number of ring atoms as said aryl group structure.

In one embodiment, R ^q is each independently halogen, -C _1-3 alkyl, -OH, -O-C _1-3 alkyl, -SH, -S-C _1-3 alkyl, -C(= O)-C _1-3 alkyl, and -S(=O) ₂ -C _1-3 alkyl. In one very specific embodiment, R ^q is selected from halogen, -CH ₃ , -OH and -OCH ₃ .

In one embodiment, L ¹ is absent.

式中、ペイロードは、水素、及び少なくとも１つのキラル中心を含む小分子化合物から選択され、
Ｌ^１は上記で定義した通りである。 In one embodiment, portion 2 has a structure of formula (V-1) below,

wherein the payload is selected from hydrogen and a small molecule compound containing at least one chiral center;
L ¹ is as defined above.

In one embodiment, the small molecule is an enzyme inhibitor, an enzyme activator, a receptor modulator (e.g., an agonist or an antagonist), a toxin (e.g., a cytotoxin), a glycan, a PEG moiety, a radionuclide (e.g., ²²⁵ Ac , ²¹¹ At, ^212Bi , ^213Bi , ^67Ga , ^123I , ^124I , 125I, ^131I , ^111In , ^177Lu , ^191mOs , ^195mPt , ^186Re , ^188Re , ^{119Sb , 153 Sm} , ^99m Tc ^{. _} protein tags, biologically active peptides, protein toxins and enzymes), low molecular weight peptidomimetics, low molecular weight antibodies (eg nanobodies) and antibody fragments of <700 Da, <600 Da, or <500 Da.

In one embodiment, moiety 2 does not include ionizable acidic or ionizable basic groups. In another embodiment, moiety 2 includes a number of ionizable acidic groups and ionizable basic groups, and under the chromatographic conditions used herein, the total charge of moiety 2 is about zero. be. Ionizable acidic groups include, but are not limited to, carboxyl groups, sulfinic acid groups, sulfonic acid groups, phosphinic acid groups, phosphonic acid groups, and the like, particularly carboxyl groups. Ionizable basic groups include, but are not limited to, amino groups, amines, etc., especially amino groups. In an alternative embodiment, the payload does not include ionizable carboxyl groups or ionizable amino groups. In another alternative embodiment, the payload includes an equal number of ionizable carboxyl groups and ionizable amino groups.

式中、毒素は、１つ又は複数のキラル中心を含む細胞毒素部分であり、Ｌ^１は上記で定義した通りである。 In one embodiment, portion 2 has a structure of the following formula (V-1-1),

where the toxin is a cytotoxin moiety containing one or more chiral centers and L ¹ is as defined above.

In one embodiment, L ¹ is absent or selected from C _1-10 alkylene groups, wherein one or more (-CH ₂ -) structures in the alkyl group are optionally replaced by oxo (=O). will be replaced with

In a preferred embodiment, the cytotoxin is a taxane, maytansinoids, auristatin, epothilones, combretastatin A-4 phosphate, combretastatin A-4 ( combretastatin A-4) and its derivatives, indole-sulfonamides, vinblastines such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine , vinglycinate , anhy-drovinblastine, dolastatin 10 and its analogues, halichondrin B, eribulin, indole-3-oxamides, podophyllotoxins, 7-diethylamino-3-(2) '-Benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide. In another embodiment, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors, such as camptothecins and their derivatives, mitoxantrone, mitoguazone, and the like. In a preferred embodiment, the cytotoxin is, for example, chlorambucil, chlornafadine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novenbitin, phenamet, fenesterine, prednimustine, trofosfamide. , uracil mustard and the like. In a further preferred embodiment, the cytotoxin is selected from the group consisting of nitrosoureas such as, for example, carmustine, flubenzuron, formoterol, lomustine, nimustine, rhamustine. In one embodiment, the cytotoxin is selected from the group consisting of aziridines. In one preferred embodiment, the cytotoxin is selected from the group consisting of benzodopa, carbocone, metredepa, and uredepa. In one embodiment, the cytotoxin is selected from the group consisting of anti-tumor antibiotics. In one preferred embodiment, the cytotoxin is selected from the group consisting of enediyne antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of dynemicin, esperamicin, neocarzinostatin, and aclacinomycin. In another preferred embodiment, the cytotoxin is actinomycin, anthramycin, bleomycins, actinomycin C, carabicin, carminomycin, cardinophilin, carminomycin, actinomycin D, daunorubicin, detruubicin, adriamycin, epirubicin, esorubicin, selected from the group consisting of idarubicin, marcellomycin, mitomycins, nogaramycin, olibomycin, pepromycin, pepromycin, puromycin, iron adriamycin, rhodorubicin, rufochromomycin, streptozocin, dinostatin, and zorubicin. In a further preferred embodiment, the cytotoxin is selected from the group consisting of trichothecenes. In a more preferred embodiment, the cytotoxin is selected from the group consisting of T-2 toxin, verracurin A, basilocporin A, and anguidine. In one embodiment, the cytotoxin is selected from the group consisting of anti-tumor amino acid derivatives. In one preferred embodiment, the cytotoxin is selected from the group consisting of ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin is selected from the group consisting of folic acid analogs. In one preferred embodiment, the cytotoxin is selected from the group consisting of dimethylfolate, methotrexate, pteropterin, trimetrexate, and edatrexate. In one embodiment, the cytotoxin is selected from the group consisting of purine analogs. In one preferred embodiment, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, thiamipurine, and thioguanine. In further embodiments, the cytotoxin is selected from the group consisting of pyrimidine analogs. In one preferred embodiment, the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, and floxuridine. In one embodiment, the cytotoxin is selected from androgens. In one preferred embodiment, the cytotoxin is selected from the group consisting of carsterone, dromostanolone propionate, epithiostanol, mepithiostane, and testolactone. In another embodiment, the cytotoxin is selected from the group consisting of anti-adrenal drugs. In one preferred embodiment, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, and trilostane. In one embodiment, the cytotoxin is selected from the group consisting of anti-androgens. In one preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprorelin acetate, and goserelin. In further embodiments, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors. In one particular embodiment, the cytotoxin is selected from the group consisting of vinblastines, colchicines, taxanes, auristatins, and maytansinoids.

In one particular embodiment, the cytotoxin is a maytansinoid, such as DM1. Note that when using a cytotoxin containing a thiol group moiety, the thiol group moiety can react with a maleimide moiety to form a thiosuccinimide, e.g., a maytansinoid, e.g. It should be noted that it is possible to connect directly to It is understood that in this case, the payload represents the remainder of the cytotoxin molecule excluding the thiol group moiety, since in some embodiments the payload and the thiol group jointly constitute the cytotoxin. It is.

In one embodiment, each portion 3 of the four compounds in mixture 1 is the same and the molecular weight of portion 3 is 1900 Da or less.

In one embodiment, the molecular weight of portion 3 is 1800 Da or less, 1700 Da or less, preferably 1600 Da or less, 1500 Da or less, 1400 Da or less, 1300 Da or less, 1200 Da or less, 1100 Da or less, 1000 Da, 900 Da or less, 800 Da or less, 700 Da or less , 600 Da or less, 500 Da or less, 400 Da or less, 300 Da or less, or 200 Da or less. In one particular embodiment, the molecular weight of moiety 3 is about 100 to about 2000 Da, preferably about 100 to about 1500 Da, about 100 to about 1000 Da, about 200 to about 1000 Da, or about 200 to about 600 Da.

式中、Ｌ^２は結合であるか、又はＣ_１－１０アルキレン基、Ｃ_３－１０シクロアルキレン基、Ｃ_６－１０アリーレン基、４～１０員ヘテロシクリレン基、５～１０員ヘテロアリーレン基、－ＮＨ－、－（ＣＯ）－、－ＮＨ（ＣＯ）－、及び－（ＣＯ）ＮＨ－から選択される１つ又は複数の二価基であり、
Ｍは、（１）１～２０個のアミノ酸を含むアミノ酸配列１と、（２）「－（Ｃ_２Ｈ_４－Ｏ）_ｉ－」構造を含み、ｉが１～１００の整数である、任意に存在するポリエチレングリコール（ＰＥＧ）部分と、（３）切断可能な配列１、スペーサーＳｐ１及びそれらの組合せから選択される二価の任意に存在する基Ｙと、を含み、
前記切断可能な配列は、酵素によって切断可能なアミノ酸配列２を含み、前記切断可能な配列は１～１０個のアミノ酸を含み、
Ｓｐ１は、１～２０個のアミノ酸を含むスペーサー配列、ＰＡＢ及びそれらの組合せから選択される。 In one particular embodiment, moiety 3 has the structure of formula (VI):

In the formula, L ² is a bond, or a C _1-10 alkylene group, a C _3-10 cycloalkylene group, a C _6-10 arylene group, a 4- to 10-membered heterocyclylene group, a 5- to 10-membered heteroarylene group , -NH-, -(CO)-, -NH(CO)-, and -(CO)NH-,
M is an arbitrary sequence containing (1) an amino acid sequence 1 containing 1 to 20 amino acids and (2) a "-(C ₂ H ₄ -O) _i -" structure, where i is an integer of 1 to 100. (3) a divalent optionally present group Y selected from cleavable sequence 1, spacer Sp1, and combinations thereof;
the cleavable sequence comprises an enzyme-cleavable amino acid sequence 2, the cleavable sequence comprises 1 to 10 amino acids,
Sp1 is selected from spacer sequences containing 1 to 20 amino acids, PAB and combinations thereof.

In one embodiment, amino acid sequence 1 comprises a ligase recognition motif, ie a ligase donor substrate recognition motif or a ligase acceptor substrate recognition motif. In one embodiment, the ligase is a transpeptidase. In one preferred embodiment, the ligase is a sortase. In one embodiment, the sortase is selected from sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), sortase F (SrtF), and combinations thereof. Ru. In one embodiment, the ligase is SrtA. In another specific embodiment, the ligase recognition motif is selected from LPXTG, where X can be any single amino acid, natural or non-natural. In one particular embodiment, the ligase recognition motif is LPETG. In a further specific embodiment, the ligase recognition motif is G _n , where G is glycine (Gly) and n is an integer from 3 to 10.

In one embodiment, i is 1-10.

In one embodiment, Y is a bond or selected from cleavable sequences, spacers Sp1, and combinations thereof. In one particular embodiment, Y is a bond. In one embodiment, amino acid sequence 2 can be recognized as an enzyme substrate and can be cleaved by the enzyme. In one particular embodiment, amino acid sequence 2 can be enzymatically cleaved in the lysosome of the cell. In another specific embodiment, amino acid sequence 2 can be cleaved by a protease, especially a cathepsin. In a further specific embodiment, amino acid sequence 2 is cleavable by glutaminase. In one embodiment, amino acid sequence 2 is selected from cathepsin restriction sites, glutaminase restriction sites, and combinations thereof. In one embodiment, the cleavable sequence is selected from Phe-Lys, Val-Cit, Val-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu and combinations thereof.

In one embodiment, Y is a bond or selected from spacer Sp1. In another embodiment, Sp1 is a spacer sequence comprising 1-10, preferably 1-6, more preferably 1-4 amino acids. In one particular embodiment, Sp1 is Leu. In another specific embodiment, Sp1 is Gln. In one embodiment, Sp1 is PAB. In further embodiments, Y is selected from Phe-Lys-PAB, Val-Cit-PAB and Val-Lys-PAB.

In one embodiment, the amino acids included in Y can be natural or non-natural. In one particular embodiment, Y is a bond or is the amino acid sequence 3. Amino acid sequence 3 contains 1 to 30 natural or unnatural amino acids, each independently the same or different. Amino acid sequence 3 is selected from cleavable sequence 1 containing 1 to 10 amino acids, spacer Sp1 containing 1 to 20 amino acids, and combinations thereof.

In one embodiment, M is selected from lysine, oligomeric glycine, oligomeric alanine, oligomeric glycine/alanine mixtures with a degree of polymerization of 3 to 10, and combinations thereof.

In one embodiment, the pKa of portion 3 is 7 or more and 12 or less. In preferred embodiments, the pKa of portion 3 is from about 8 to about 12, preferably from about 9 to about 11, such as about 9.0, about 9.1, about 9.2, about 9.3, about 9 .4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.1, about 10.2, about 10.3, about 10 .4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9, about 11.0, especially about 10.0.

In one particular embodiment, M is L(G) _n , where G is glycine (Gly) and n is an integer from 3 to 10, especially 3. In another specific embodiment, M is G _n . In one embodiment, the C-terminus of M is linked to L ² and M has an ionizable amino group. In one particular embodiment, M has only one ionizable group and is an ionizable amino group. An ionizable amino group can be ionized to the group -NH ₃ ⁺ . The ionized form of the amino group -NH ₃ ⁺ can optionally interact with solvent molecules or chemicals, said chemicals having a negative charge (+) or a partial negative charge (δ+).

In an alternative embodiment, M is LPXTGJ, where X can be any single amino acid, natural or non-natural, and J is absent or an amino acid fragment containing from 1 to 10 amino acids. and optionally has a tag. In one embodiment, J is absent. In another embodiment, J is an amino acid fragment containing 1-10 amino acids, in which each amino acid is independently any natural or unnatural amino acid. In another embodiment, J is G _m , where m is an integer from 1 to 10. In further specific embodiments, M is LPETG. In another specific embodiment, M is LPETGG. In one embodiment, the N-terminus of M is linked to L ² and M has a free C-terminal carboxyl group. In one particular embodiment, M has only one ionizable group and is an ionizable carboxyl group. Ionizable carboxyl groups can be ionized to the group -COO ^- . The ionized form of the carboxyl group -COO ^- can optionally interact with solvent molecules or chemicals, said chemicals having a positive or partially positive charge (δ+).

式中、ｎは３～１０の整数であり、ｘは、水素、ＯＨ、ＮＨ_２、１～１０個のアミノ酸を含むアミノ酸フラグメント、及び１～１０個のヌクレオチドを含むヌクレオチドフラグメントから選択される。 In one embodiment, the structure of formula (VI) has the structure of formula (VI-1) below,

where n is an integer from 3 to 10 and x is selected from hydrogen, OH, NH ₂ , amino acid fragments containing 1 to 10 amino acids, and nucleotide fragments containing 1 to 10 nucleotides.

In one embodiment, n is 3. In one preferred embodiment, x is selected from OH, NH ₂ , an amino acid fragment containing 1 to 10 amino acids. In a more preferred embodiment, x is selected from OH, _NH2 and GIy. In one particular embodiment, x is _NH2 .

In one embodiment, L ¹ and L ² are each independently selected from a bond and a C _1-10 alkyl group, and one or more (-CH ₂ -) structures in the alkyl group are oxo (=O ) may be optionally replaced by

から選択され、部分３中のＬ^２は結合である。 In one embodiment, L ¹ in portion 2 is

and L ² in portion 3 is a bond.

から選択される。 In another embodiment, L ¹ in portion 2 is a bond and L ² in portion 3 is

selected from.

式中、ペイロードは式（Ｖ－１）で定義された通りであり、Ｍは式（ＶＩ）で定義された通りである。 In one embodiment, the four compounds contained in mixture 1 each independently have a structure selected from the following formulas (1) and (1'),

where the payload is as defined in equation (V-1) and M is as defined in equation (VI).

式中、ペイロードは式（Ｖ－１）で定義された通りであり、Ｙは式（ＶＩ）で定義された通りであり、ｎとｘは式（ＶＩ－１）で定義された通りである。 In one embodiment, the four compounds contained in Mixture 1 each independently have a structure selected from the following formulas (2) and (2'),

where the payload is as defined in equation (V-1), Y is as defined in equation (VI), and n and x are as defined in equation (VI-1). .

式中、ペイロードは式（Ｖ－１）で定義された通りであり、Ｍは式（ＶＩ）で定義された通りである。 In one particular embodiment, the compound of formula (1) and the compound of formula (1′) are products of a ring-opening reaction of a thiosuccinimide group in the following formula (3),

where the payload is as defined in equation (V-1) and M is as defined in equation (VI).

式中、ペイロードは式（Ｖ－１）で定義された通りであり、ｎとｘは式（ＶＩ－１）で定義された通りである。 In one particular embodiment, the compound of formula (2) and the compound of formula (2′) are products of a ring-opening reaction of a thiosuccinimide group in the following formula (4),

where the payload is as defined in equation (V-1), and n and x are as defined in equation (VI-1).

The ring-opening reaction of the thiosuccinimide group can be carried out by any method known in the art, for example a method for the ring-opening reaction can be found in WO2015165413A1.

In one particular embodiment, M has an ionizable amino group, Acidic Agent 1 is AcOH, and the amount of Acidic Agent 1 is from about 0.01% to about 1% based on the total volume of Eluent A. %, preferably about 0.05% to about 0.5%, more preferably about 0.1% to about 0.5%, more preferably about 0.1% to about 0.3%, especially 0. .3% and no acidic agent 2 is present.

In another specific embodiment, M has an ionizable amino group, acidic agent 3 is TFA, and the amount of acidic agent 3 is from about 0.01% to about 0.01% based on the total volume of eluent C. about 1%, preferably about 0.05% to about 0.5%, more preferably about 0.05% to about 0.3%, especially 0.1%, and the acidic agent 4 is not present. , or about 0.01% to about 1%, preferably about 0.05% to about 0.5%, more preferably about 0.05% to about 0.3%, based on the total volume of eluent D. In particular it is present in an amount of 0.1%.

In one embodiment, mixture 1 is a reaction mixture resulting from a ring-opening reaction of thiosuccinimide groups resulting in the ring-opened thiosuccinimide structure of moiety 1.

In an alternative embodiment, M comprises one or more primary amino groups, and each acidic agent 1-4 forms an ion pair with one or more primary amino groups comprised in M. can do. In another alternative embodiment, acidic agents 1-4 are independently selected from AcOH, L-TA and TFA. In one particularly specific embodiment, acidic agent 1 is AcOH or L-TA, preferably AcOH. In another particularly specific embodiment, acidic agent 3 is TFA.

式中、Ｒ^１は水素及びＣ_１－１０アルキル基から選択され、各Ｒ^１のキラル立体配置は式（ＶＩＩ）～（Ｘ）において同じである。 In an alternative embodiment, moiety 1 is further substituted by R ¹ and moiety 1 has a structure selected from formulas (VII) to (X) below;

where R ¹ is selected from hydrogen and a C _1-10 alkyl group, and the chiral configuration of each R ¹ is the same in formulas (VII) to (X).

In one embodiment, the chromatography in step (2) is performed on reverse phase HPLC. In an alternative embodiment, the chromatography in step (4) is carried out by a method selected from normal pressure chromatography, medium pressure chromatography and high pressure chromatography. In another embodiment, the reversed phase chromatography in step (4) is performed on reversed phase HPLC.

式中、ｘは－ＯＨ又は－ＮＨ_２である。具体的な一実施形態において、ｘは－ＮＨ_２であり、式（ｉ）～（ｉｖ）の化合物は式（ｉ－１）～（ｉｖ－１）の構造を有する。 In one particular embodiment, each of the four compounds included in mixture 1 has a structure of formulas (i) to (iv),

In the formula, x is -OH or _-NH2 . In one specific embodiment, x is -NH ₂ and the compounds of formulas (i) to (iv) have the structures of formulas (i-1) to (iv-1).

In one particular embodiment, compounds (i) and (ii) are obtained as separate products in step (2) and compounds (iii) and (iv) are obtained as separate products in step (4). .

In another specific embodiment, in step (2) compounds (i) and (ii) are obtained as separate products and compounds (iii) and (iv) are obtained as mixture 2.

式中、ｘは－ＯＨ又は－ＮＨ_２である。 In one embodiment, mixture 1 is a reaction mixture obtained by ring opening reaction of thiosuccinimide groups in the following compounds:

In the formula, x is -OH or _-NH2 .

In one particular embodiment, for the reversed phase chromatography separation range of step (2),
B is a slope of about 43% to about 53%, the remainder is A, and the time is 10 to 100 minutes, preferably 30 to 50 minutes.

In one particular embodiment, for the reversed phase chromatography separation range of step (2),
B is a slope of about 43% to about 48%, the remainder is A, and the time is 10 to 100 minutes, preferably 48 minutes.

In one particular embodiment, the column temperature for reverse phase chromatography in step (2) is about 10 to about 40°C, preferably about 20 to about 30°C. In another specific embodiment, the loading amount for reverse phase column chromatography is 20 g/Kg or less, 19 g/Kg or less, 18 g/Kg or less, 17 g/Kg or less, 16 g/Kg, 15 g/Kg or less, 14 g/Kg or less. kg or less, 13 g/Kg or less, 12 g/Kg or less, 11 g/Kg or less, 10 g/Kg or less, 9 g/Kg or less, 8 g/Kg or less, 7 g/Kg or less, and 6 g/Kg or less. In one preferred embodiment, the loading amount for reverse phase column chromatography is 14 g/Kg or less. In a very particular embodiment, the loading amount for reverse phase column chromatography is about 0.1 g/Kg to 14 g/Kg, preferably 0.1 g/Kg to 14 g/Kg, and the mobile phase flow rate is constant. and in the range of about 100±50 mL/min to about 500±50 mL/min, particularly about 300±50 mL/min.

In another specific embodiment, for the separation range of the reversed phase chromatography in step (4),
D is a slope of about 22% to about 42%, the remainder is C, and the time is 20 to 100 minutes.

In another specific embodiment, for the separation range of the reversed phase chromatography in step (4),
D is about 22% to about 28% slope, the rest is C, time is 44 minutes.

In one particular embodiment, the column temperature for reverse phase chromatography in step (2) is about 10 to 40°C, preferably about 20 to about 30°C. In another specific embodiment, the loading amount for reverse phase column chromatography is 20 g/Kg or less, 19 g/Kg or less, 18 g/Kg or less, 17 g/Kg or less, 16 g/Kg or less, 15 g/Kg or less, 14 g /Kg or less, 13g/Kg or less, 12g/Kg or less, 11g/Kg or less, 10g/Kg or less, 9g/Kg or less, 8g/Kg or less, 7g/Kg or less, and 6g/Kg or less. In one preferred embodiment, the loading amount for reverse phase column chromatography is 14 g/Kg or less. In a very particular embodiment, the loading amount for reverse phase column chromatography is about 0.1 g/Kg to 14 g/Kg, preferably 0.1 g/Kg to 14 g/Kg, and the mobile phase flow rate is constant. and in the range of about 100±50 mL/min to about 500±50 mL/min, particularly about 300±50 mL/min.

Analytical Method The inventor unexpectedly discovered that the separation method provided in the first aspect of the present disclosure can be effectively applied to the analysis of the four compounds contained in Mixture 1 or Mixture 2. For example, the effectiveness of a separation process can be determined on an analytical scale with sufficient precision under substantially the same chromatographic conditions.

Accordingly, in a second aspect, the present disclosure provides a method for analyzing one or more compounds in a mixture 1, said mixture 1 comprising four compounds, each of the four compounds in the mixture Includes part 1, part 2 and part 3.

The method comprises applying step (2) on an analytical scale, where part 1, part 2, part 3 and step (2) are as defined above.

In one embodiment, the method further comprises applying step (4) on an analytical scale, where step (4) is as defined above.

In a third aspect, the present disclosure provides a method for analyzing one or more compounds in a mixture 2, said mixture 2 comprising two compounds, each of the two compounds in the mixture being part 1 , including part 2 and part 3.

The method comprises applying step (4) on an analytical scale, where part 1, part 2, part 3 and step (4) are as defined above.

In one embodiment, the robustness of the chromatographic conditions of the analytical process is optionally studied. In one embodiment, the chromatographic conditions of the analytical process are adjusted based on the results of system applicability measurements. Each analytical process can be independently used as a process control method for one or more preparative processes. In one embodiment, the preparative and analytical processes have the same chromatographic conditions. In another embodiment, the chromatographic conditions used in the preparative process and the chromatographic conditions used in the analytical process differ only in the particle size and/or pore size of the stationary phase. In one embodiment, both the preparative and analytical processes use gradient elution. In another embodiment, the preparative process uses gradient elution and the analytical process uses isocratic elution.

In one particular embodiment, the loading volume of the analytical process is 1 ml or less. In another specific embodiment, the loading volume for the analytical process is 1-300 μL, such as 5-100 μL. In a very specific embodiment, the flow rate of the mobile phase is less than or equal to 1 mL/min, for example the flow rate can be between 0.5 and 1 mL/min, especially 0.7 mL/min, 0.8 mL/min, It is 0.9 mL/min or 1.0 mL/min.

式中、
ペイロード、Ｌ^１、Ｌ^２及びＭは上記で定義した通りである。 Isomeric Compounds of the Disclosure Payload-Linker Intermediate In a fourth aspect, the present disclosure provides a compound having the structure of any one of the following formulas (XI) to (XIV),

During the ceremony,
The payloads, L ¹ , L ² and M are as defined above.

であり、Ｍは

であり、式（ＸＩ）～（ＸＩＶ）はそれぞれ、下記式（ＸＩ－１）～（ＸＩＶ－１）の構造を有し、

式中、
ペイロード、Ｙ、ｎ及びｘは上記で定義した通りである。 In one embodiment, L ¹ is absent and L ² is

and M is

and formulas (XI) to (XIV) have the structures of the following formulas (XI-1) to (XIV-1), respectively,

During the ceremony,
Payload, Y, n and x are as defined above.

Depending on the position of the thiol group, formulas (XI) and (XII) are referred to as β, and the configurations of formulas (XIII) and (XIV) are referred to as α.

In one embodiment, Y is a bond, n is 3, and formulas (XI-1) to (XIV-1) are of the following formulas (XI-1-1) to (XIV-1-1), respectively. Has a structure.

In one particular embodiment, the payload is a cytotoxic moiety containing one or more chiral centers, preferably a maytansinoid, more preferably DM1. In one embodiment, the payload is DM1 and formulas (XI-1-1) to (XIV-1-1) represent the structures of isomeric compounds (i) to (iv), the definitions of which are as above. .

In a fifth aspect, the disclosure provides a mixture comprising two compounds, each compound therein having a structure of any one of formulas (XI) to (XIV), provided that said two compounds are provided that they have different structures. In one embodiment, the mixture comprises two compounds having the structures of formula (XI) and formula (XII), respectively. In another embodiment, the mixture comprises two compounds having the structures of formula (XIII) and formula (XIV), respectively. In one embodiment, the mixture includes two compounds having the structures of formula (XI-1) and formula (XII-1), respectively. In another embodiment, the mixture comprises two compounds having the structures of formula (XIII-1) and formula (XIV-1), respectively. In one embodiment, the mixture includes two compounds having the structures of formula (XI-1-1) and formula (XII-1-1), respectively. In another embodiment, the mixture comprises two compounds having the structures of formula (XIII-1-1) and formula (XIV-1-1), respectively. In one particular embodiment, the mixture comprises two compounds, each having the structure of formula (i) and formula (ii). In another embodiment, the mixture comprises two compounds, each having the structure of formula (iii) and formula (iv).

In the following, the mixture of compounds (iii) and (iv) is referred to as α intermediate or α isomer.

In some embodiments, the compounds provided in the present disclosure can be described as <1> to <10> below.

式中、
ペイロードは、１つ又は複数のキラル中心を含む細胞毒素部分であり、
Ｌ^１は存在しないか、又はＣ_１－１０アルキレン基、Ｃ_３－１０シクロアルキレン基、Ｃ_６－_１０アリーレン基、４～１０員ヘテロシクリレン基、５～１０員ヘテロアリーレン基、－ＮＨ－、－（ＣＯ）－、－ＮＨ（ＣＯ）－、及び－（ＣＯ）ＮＨ－から選択される１つ又は複数の二価基であり、
Ｌ^２は結合であるか、又はＣ_１－１０アルキレン基、Ｃ_３－１０シクロアルキレン基、Ｃ_６－_１０アリーレン基、４～１０員ヘテロシクリレン基、５～１０員ヘテロアリーレン基、－ＮＨ－、－（ＣＯ）－、－ＮＨ（ＣＯ）－、及び－（ＣＯ）ＮＨ－から選択される１つ又は複数の二価基であり、
Ｍはリガーゼ認識モチーフを含み、
前記化合物は、主に、例えば純粋又は実質的に純粋な異性体形態であり、例えば他の異性体形態を実質的に含まず、例えば９０％超、例えば９５％超、例えば９８％超、例えば少なくとも９９％の純度を有する、前記化合物。 <1> A compound of formula (XI), (XII), (XIII) or (XIV),

During the ceremony,
The payload is a cytotoxin moiety containing one or more chiral centers;
L ¹ is absent or C _1-10 alkylene group, C _3-10 cycloalkylene group, C _6-10 arylene group, _4-10 membered heterocyclylene group, 5-10 membered heteroarylene group, -NH- , -(CO)-, -NH(CO)-, and -(CO)NH-,
L ² is a bond, or a C _1-10 alkylene group, a C _3-10 cycloalkylene group, a C _6-10 arylene group, a _4-10 membered heterocyclylene group, a 5-10 membered heteroarylene group, -NH one or more divalent groups selected from -, -(CO)-, -NH(CO)-, and -(CO)NH-,
M contains a ligase recognition motif;
The compound may be primarily in pure or substantially pure isomeric form, e.g. substantially free of other isomeric forms, e.g. greater than 90%, e.g. greater than 95%, e.g. greater than 98%, e.g. Said compound having a purity of at least 99%.

<2> The compound according to <1>, which is a compound of formula (XIII) or (XIV).

<3> The ligase recognition motif is (a) LPXTG, for example, LPETG, where X is any amino acid residue, and (b) G n, where G is glycine (Gly) and _n is an integer from 3 to 10. The compound according to <1> or <2>, selected from.

<4> The compound according to <3>, wherein M is H-Gly-Gly-Gly-Lys-NH ₂ and M is linked to L ² via the ε-amino group on lysine.

から選択される、＜１＞～＜４＞のいずれか１項に記載の化合物。 <5> L ¹ does not exist, and L ² does not exist.

The compound according to any one of <1> to <4>, selected from

であり、Ｍは

であり、
式中、
Ｙは結合であるか又は１～２０個のアミノ酸のスペーサー配列であり、
ｎは３～１０の整数、例えば３であり、
ｘは、水素、－ＯＨ、－ＮＨ_２、１～１０個のアミノ酸を含むアミノ酸フラグメント、及び１～１０個のヌクレオチドを含むヌクレオチドフラグメントから選択され、例えば－ＯＨ、－ＮＨ_２、及び１～１０個のアミノ酸を含むアミノ酸フラグメントから選択され、例えば－ＯＨ、－ＮＨ_２及びＧｌｙから選択され、例えば－ＮＨ_２である、＜１＞～＜４＞のいずれか１項に記載の化合物。 <6> L ¹ does not exist, L ² is

and M is

and
During the ceremony,
Y is a bond or a spacer sequence of 1 to 20 amino acids;
n is an integer from 3 to 10, for example 3;
x is selected from hydrogen, -OH, _-NH2 , amino acid fragments containing 1 to 10 amino acids, and nucleotide fragments containing 1 to 10 nucleotides, such as -OH, _-NH2 , and 1 to 10 The compound according to any one of <1> to <4>, wherein the compound is selected from amino acid fragments comprising -OH, -NH ₂ and Gly, for example -NH ₂ .

<7> The compound according to any one of <1> to <6>, wherein the cytotoxin contains a thiol group moiety that can react with a maleimide moiety.

である、＜１＞～＜７＞のいずれか１項に記載の化合物。 <8> The cytotoxin is a maytansinoid,
For example, DM1

The compound according to any one of <1> to <7>, which is

式中、ｘは－ＯＨ又は－ＮＨ_２である、＜１＞～＜８＞のいずれか１項に記載の化合物。 <9> selected from compounds of formula (i), (ii), (iii) or (iv),

The compound according to any one of <1> to <8>, wherein x is -OH or -NH ₂ .

<10> The compound according to <9>, which is a compound of formula (iii) or (iv).

In one aspect, the present disclosure provides a method for preparing a compound of formula (XI), (XII), (XIII) or (XIV), as in the following <17> to <18>.

<17> A method for preparing a compound of formula (XI), (XII), (XIII) or (XIV), comprising:
(i) synthesizing a mixture of compounds of formulas (XI), (XII), (XIII) and (XIV);
(ii) subjecting the mixture to chromatography to obtain a target compound,
said step in which each of said target compounds is obtained as a separate product or the two target compounds are obtained as a second mixture;
(iii) optionally collecting an eluate comprising a third mixture collected in step (ii), said third mixture containing one or more additional target compounds different from the target compound separated in step (2); chromatography of the collected eluate containing a target compound to separate said additional target compound.

<18> The method according to <17>, wherein the chromatography is reversed phase chromatography.

Antibody Drug Conjugates A compound of formula (XI), (XII), (XIII) or (XIV) can be conjugated with a biomolecule containing a ligase recognition motif to form a bioconjugate, wherein the The ligase recognition motif contained in the biomolecule corresponds to the ligase recognition motif contained in amino acid sequence 1 in structure M of the compound of formula (XI), (XII), (XIII) or (XIV).

In one embodiment, the compound of formula (XI), (XII), (XIII) or (XIV) comprises a ligase acceptor substrate recognition motif GGG and the biomolecule comprises a ligase donor substrate recognition motif LPXTG.

式中、
Ａは、リガーゼ供与体基質認識モチーフ又はリガーゼ受容体基質認識モチーフを有するように任意に修飾された抗体又はその抗原結合フラグメントであり、
Ｌ^３は存在しないか、又は、（１）１～２０個のアミノ酸を含むアミノ酸配列４と、（２）「－（Ｃ_２Ｈ_４－Ｏ）_ｊ－」構造を含み、ｊが１～１００の整数である任意に存在するＰＥＧフラグメントと、（３）切断可能な配列２、２～１００個のアミノ酸を含むスペーサーＳｐ２及びそれらの組合せから選択される二価の任意に存在する基Ｕと、を含み、
切断可能な配列２は、酵素によって切断可能なアミノ酸配列５を含み、切断可能な配列２は１～１０個のアミノ酸を含み、
ペイロード、Ｙ、ｎ及びｘは上記で定義した通りである。 In a sixth aspect, the disclosure provides an antibody-drug conjugate (ADC) prepared using a compound of formula (XI), (XII), (XIII) or (XIV) and an antibody or antigen-binding fragment thereof. . Preparation of the ADC can be carried out by any method known in the art, for example by coupling the ligase recognition motif contained in structure M to the ligase recognition motif contained in the antibody or antigen-binding fragment thereof. be able to. The obtained ADC further comprises an isomeric ring-opened thiosuccinimide group in a compound such as formula (XI), (XII), (XIII) or (XIV), and the obtained ADC has the following formula (XVII- 1) to have any one structure of (XX-1),

During the ceremony,
A is an antibody or antigen-binding fragment thereof optionally modified to have a ligase donor substrate recognition motif or a ligase acceptor substrate recognition motif;
L ³ is absent or contains (1) an amino acid sequence 4 containing 1 to 20 amino acids and (2) a "-(C ₂ H ₄ -O) _j -" structure, where j is 1 to 100 (3) a divalent optionally present group U selected from the cleavable sequence 2, a spacer Sp2 comprising from 2 to 100 amino acids, and combinations thereof; including;
Cleavable sequence 2 comprises an enzymatically cleavable amino acid sequence 5, cleavable sequence 2 comprises 1 to 10 amino acids,
Payload, Y, n and x are as defined above.

In one embodiment, j is an integer from 1 to 10.

In one embodiment, Sp2 is a spacer sequence containing 2-20 amino acids. In one particular embodiment, Sp2 is a spacer sequence selected from GA, GGGS and GGGGSGGGGS, especially GA.

In one embodiment, the introduction position of the ligase substrate recognition motif is not limited; for example, the introduction position can be, but is not limited to, the C-terminus or N-terminus of the antibody heavy chain or light chain.

The light chain of the antibody or antigen-binding fragment thereof can be wild-type (LC), C-terminally modified light chain (LCCT) modified by direct introduction of a ligase recognition motif LPXTG, a short peptide spacer and a ligase donor substrate recognition and a C-terminally modified light chain (LCCT _L ) modified by the introduction of the motif LPXTG. The heavy chains of antibodies or antigen-binding fragments thereof can be wild-type (HC), C-terminally modified heavy chains (HCCT) modified by direct introduction of a ligase recognition motif LPXTG, a short peptide spacer and a ligase donor substrate recognition motif. C-terminally modified heavy chain (HCCT _L ) modified by the introduction of LPXTG. X can be any single natural or non-natural amino acid. When z in the compound of formula (IV) is 1 or 2, the combination of the above heavy and light chains can form eight preferred antibody molecules. Sortase-mediated ligation forms a new amide bond between the C-terminal sorting sequence LPXTG and the N-terminal GGG, where the conjugation reaction first creates a peptide bond between the threonine and glycine residues. This is done by cutting and then ligating LPXT to GGG.

In a preferred embodiment, the light chain of the antibody or antigen-binding fragment thereof is a wild type (LC), an N-terminally modified light chain (LCNT) modified by direct introduction of a ligase recognition motif GGG, a short peptide spacer and and an N-terminally modified light chain (LCNT _L ) modified by the introduction of the ligase receptor substrate recognition motif GGG. The heavy chains of antibodies or antigen-binding fragments thereof can be wild-type (HC), N-terminally modified heavy chains (HCNT) modified by direct introduction of a ligase recognition motif GGG, a short peptide spacer and a ligase receptor substrate recognition motif. N-terminally modified heavy chains modified by the introduction of GGG (HCNT _L ).

式中、
ペイロード、Ａ、ｘ及びｚは上記で定義した通りである。 In one embodiment, Y is a bond, n is 3, and formulas (XVII-1) to (XX-1) are of the following formulas (XVII-1-1) to (XX-1-1), respectively. has a structure,

During the ceremony,
Payload, A, x and z are as defined above.

In one embodiment, the payload is DM1, z is 2, and formulas (XI-1) to (XIV-1) are the following formulas (XI-1-1) to (XIV-1-1), respectively: Has a structure.

式中、
Ａとｘは上記で定義した通りである。具体的な一実施形態において、ｘは－ＮＨ_２である。 In one particular embodiment, the payload is a cytotoxin moiety that includes one or more chiral centers. The cytotoxin is preferably a maytansinoid, more preferably DM1. In one embodiment, the payload is DM1, L ³ is absent, and formulas (XVII-1-1) to (XX-1-1) have the structures of compounds (v) to (viii) below, respectively. death,

During the ceremony,
A and x are as defined above. In one specific embodiment, x is _-NH2 .

In one embodiment, A is Trastuzumab, optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif. In one preferred embodiment, A is trastuzumab with a C-terminally modified light chain (LCCT _L ) modified by the introduction of a short peptide spacer and a ligase donor substrate recognition motif LPXTGJ, where J is defined above. That's right. In one embodiment, the short peptide spacer is GA. In another embodiment, the heavy chain of the antibody or antigen-binding fragment thereof is a wild-type heavy chain (HC).

In a seventh aspect, the disclosure provides a mixture comprising two compounds, each compound having a structure of any one of formulas (XVII-1) to (XX-1), with the proviso that: Provided that the two compounds have different structures. In one embodiment, the mixture comprises two compounds having the structures of formula (XVII-1) and formula (XVIII-1), respectively. In another embodiment, the mixture includes two compounds having the structures of formula (XIX-1) and formula (XX-1), respectively. In one embodiment, the mixture includes two compounds having the structures of formula (XVII-1-1) and formula (XVIII-1-1), respectively. In another embodiment, the mixture includes two compounds having the structures of formula (XIX-1-1) and formula (XX-1-1), respectively. In one particular embodiment, the mixture comprises two compounds, each having the structure of formula (v) and formula (vi). In another embodiment, the mixture comprises two compounds, each having the structure of formula (vii) and formula (viii). In the following, the mixture of compounds (vii) and (viii) is referred to as αADC.

In some embodiments, the antibody drug conjugates provided in the present disclosure can be described as <11> to <16> below.

前記化合物は、主に、例えば純粋又は実質的に純粋な異性体形態であり、例えば他の異性体形態を実質的に含まず、例えば９０％超、例えば９５％超、例えば９８％超、例えば少なくとも９９％の純度を有する、抗体薬物コンジュゲート。 An antibody drug conjugate comprising an open ring thiosuccinimide structure of formula (I), (II), (III) or (IV),

The compound may be primarily in pure or substantially pure isomeric form, e.g. substantially free of other isomeric forms, e.g. greater than 90%, e.g. greater than 95%, e.g. greater than 98%, e.g. An antibody drug conjugate having a purity of at least 99%.

<12> The antibody-drug conjugate according to <11>, which is formed by reacting the compound according to any one of <1> to <11> with an antibody in the presence of a ligase.

式中、
ペイロードとｘは上記で定義した通りであり、例えば１つ又は複数のキラル中心を含む細胞毒素部分、例えばチオール基部分を除いたメイタンシノイド分子の残りの部分、例えばチオール基部分を除いたＤＭ１の残りの部分であり、
Ａは、リガーゼ供与体基質認識モチーフ又はリガーゼ受容体基質認識モチーフを有するように任意に修飾された抗体又はその抗原結合フラグメントであり、
例えば、Ａは、ヒト上皮成長因子受容体２（ＨＥＲ２）に結合する抗体であり、例えばトラスツズマブであり、前記トラスツズマブは、リガーゼ供与体基質認識モチーフ又はリ ガーゼ受容体基質認識モチーフを有するように修飾され、
ｚは１又は２の整数である、＜１１＞又は＜１２＞に記載の抗体薬物コンジュゲート。 <13> Each has formulas (XVII-1-1) to (XX-1-1),

During the ceremony,
The payload and x are as defined above, e.g. the remainder of the maytansinoid molecule excluding the cytotoxin moiety containing one or more chiral centers, e.g. DM1 excluding the thiol moiety. is the rest of
A is an antibody or antigen-binding fragment thereof optionally modified to have a ligase donor substrate recognition motif or a ligase acceptor substrate recognition motif;
For example, A is an antibody that binds to human epidermal growth factor receptor 2 (HER2), such as trastuzumab, wherein the trastuzumab is modified to have a ligase donor substrate recognition motif or a ligase acceptor substrate recognition motif. is,
The antibody-drug conjugate according to <11> or <12>, wherein z is an integer of 1 or 2.

<14> A is trastuzumab with a C-terminally modified light chain (LCCT _L ) modified by the introduction of a short peptide spacer such as -GA- and a ligase donor substrate recognition motif LPXTGJ, where J in LPXTGJ is The antibody-drug conjugate according to <13>, which is absent or is an amino acid fragment containing 1 to 10 amino acids.

式中、ｘは－ＯＨ又は－ＮＨ_２である、＜１３＞又は＜１４＞に記載の抗体薬物コンジュゲート。 <15> The payload is DM1, ^L3 does not exist,
Formulas (XVII-1-1) to (XX-1-1) each have structures (v) to (viii),

The antibody-drug conjugate according to <13> or <14>, wherein x is -OH or -NH ₂ .

<16> The antibody-drug conjugate according to <14>, which has the structure (vii) shown in <15>.

Compound Mixtures and Antibody Drug Conjugate Mixtures In some embodiments, the alpha isomers of the present disclosure are provided in the form of mixtures, and thus, in an eighth aspect, the present disclosure provides compositions comprising the alpha isomers of the present disclosure. I will provide a. In some embodiments, the composition of the present disclosure can be described as <45> to <52> below.

式中、
ペイロードは、１つ又は複数のキラル中心を含む細胞毒素部分であり、
Ｌ^１は存在しないか、又はＣ_１－１０アルキレン基、Ｃ_３－１０シクロアルキレン基、Ｃ_６－_１０アリーレン基、４～１０員ヘテロシクリレン基、５～１０員ヘテロアリーレン基、－ＮＨ－、－（ＣＯ）－、－ＮＨ（ＣＯ）－、及び－（ＣＯ）ＮＨ－から選択される１つ又は複数の二価基であり、
Ｌ^２は結合であるか、又はＣ_１－１０アルキレン基、Ｃ_３－１０シクロアルキレン基、Ｃ_６－_１０アリーレン基、４～１０員ヘテロシクリレン基、５～１０員ヘテロアリーレン基、－ＮＨ－、－（ＣＯ）－、－ＮＨ（ＣＯ）－、及び－（ＣＯ）ＮＨ－から選択される１つ又は複数の二価基であり、
Ｍはリガーゼ認識モチーフを含み、
前記組成物は、式ＸＩの化合物又は式ＸＩＩの化合物を実質的に含まない（例えば、少なくとも９０％、少なくとも９５％、少なくとも９８％又は少なくとも９９％含まない）、前記組成物。

<45> A composition comprising a mixture of the compound of formula (XIII) and the compound of formula (XIV), for example a racemic mixture,

During the ceremony,
The payload is a cytotoxin moiety containing one or more chiral centers;
L ¹ is absent or C _1-10 alkylene group, C _3-10 cycloalkylene group, C _6-10 arylene group, _4-10 membered heterocyclylene group, 5-10 membered heteroarylene group, -NH- , -(CO)-, -NH(CO)-, and -(CO)NH-,
L ² is a bond, or C _1-10 alkylene group, C _3-10 cycloalkylene group, C _6-10 arylene group, _4-10 membered heterocyclylene group, 5-10 membered heteroarylene group, -NH one or more divalent groups selected from -, -(CO)-, -NH(CO)-, and -(CO)NH-,
M contains a ligase recognition motif;
The composition is substantially free (eg, at least 90%, at least 95%, at least 98% or at least 99% free) of a compound of Formula XI or a compound of Formula XII.

<46> The ligase recognition motif includes (a) LPXTG, for example, LPETG, where X is any amino acid residue, and (b) G n, where G is glycine (Gly) and _n is an integer from 3 to 10. The composition according to <45>, selected from the following.

<47> The composition according to <45> or <46>, wherein M is H-Gly-Gly-Gly-Lys-NH ₂ and M is linked to L ² via the ε-amino group on lysine. thing.

から選択される、＜４５＞～＜４７＞のいずれか１項に記載の組成物。 <48>L ¹ does not exist, L ² does not exist

The composition according to any one of <45> to <47>, selected from.

であり、Ｍは

であり、
式中、
Ｙは結合であるか又は１～２０個のアミノ酸のスペーサー配列であり、
ｎは３～１０の整数、例えば３であり、
ｘは、水素、－ＯＨ、－ＮＨ_２、１～１０個のアミノ酸を含むアミノ酸フラグメント、及び１～１０個のヌクレオチドを含むヌクレオチドフラグメントから選択され、例えば－ＯＨ、－ＮＨ_２、及び１～１０個のアミノ酸を含むアミノ酸フラグメントから選択され、例えば－ＯＨ、－ＮＨ_２及びＧｌｙから選択され、例えば－ＮＨ_２である、＜４５＞～＜４８＞のいずれか１項に記載の組成物。 <49> L ¹ does not exist, L ² is

and M is

and
During the ceremony,
Y is a bond or a spacer sequence of 1 to 20 amino acids;
n is an integer from 3 to 10, for example 3;
x is selected from hydrogen, -OH, _-NH2 , amino acid fragments containing 1 to 10 amino acids, and nucleotide fragments containing 1 to 10 nucleotides, such as -OH, _-NH2 , and 1 to 10 The composition according to any one of <45> to <48>, wherein the composition is selected from amino acid fragments comprising -OH, -NH ₂ and Gly, for example -NH ₂ .

<50> The composition according to any one of <45> to <49>, wherein the cytotoxin contains a thiol group moiety that can react with a maleimide moiety.

である、＜４５＞～＜５０＞のいずれか１項に記載の組成物。 <51> The cytotoxin is a maytansinoid, for example DM1

The composition according to any one of <45> to <50>.

<52> The compound of formula (iii) and the compound of formula (iv), which is a mixture of the compound of formula (iii) and the compound of formula (iv), which does not substantially contain the compound of formula (i) or the compound of formula (ii) iv) A mixture of compounds.

In a ninth aspect, the disclosure provides an antibody drug conjugate composition comprising a mixture of α-ADCs of the disclosure. In some embodiments, the antibody drug conjugate composition of the present disclosure can be described as <53> to <57> below.

前記組成物は、式（Ｉ）の化合物又は式（ＩＩ）の化合物を実質的に含まない（例えば、少なくとも９０％、少なくとも９５％、少なくとも９８％又は少なくとも９９％含まない）、前記抗体薬物コンジュゲート組成物。

<53> An antibody-drug conjugate comprising a mixture, for example a racemic mixture, of an antibody-drug conjugate containing an open-ring thiosuccinimide structure of formula (III) and an antibody-drug conjugate containing an open-ring thiosuccinimide structure of formula (IV) A composition,

The composition is substantially free (e.g., at least 90%, at least 95%, at least 98% or at least 99% free) of a compound of formula (I) or a compound of formula (II). Gate composition.

<54> The antibody-drug conjugate composition according to <53>, which is formed by reacting the composition according to any one of <45> to <52> with an antibody in the presence of a ligase.

式中、
ペイロードは上記で定義した通りであり、例えば１つ又は複数のキラル中心を含む細胞毒素部分、例えばチオール基部分を除いたメイタンシノイド分子の残りの部分、例えばチオール基部分を除いたＤＭ１の残りの部分であり、
Ａは、リガーゼ供与体基質認識モチーフ又はリガーゼ受容体基質認識モチーフを有するように任意に修飾された抗体又はその抗原結合フラグメントであり、例えば、Ａは、ヒト上皮成長因子受容体２（ＨＥＲ２）に結合する抗体であり、例えばトラスツズマブであり、前記トラスツズマブは、リガーゼ供与体基質認識モチーフ又はリガーゼ受容体基質認識モチーフを有するように修飾され、
ｚは１又は２の整数である、＜５２＞、＜５３＞又は＜５４＞に記載の抗体薬物コンジュゲート組成物。 <55> A mixture of a compound having the formula (XIX-1-1) and a compound having the formula (XX-1-1),

During the ceremony,
The payload is as defined above, e.g. a cytotoxin moiety containing one or more chiral centers, e.g. the remainder of the maytansinoid molecule excluding the thiol moiety, e.g. the remainder of DM1 without the thiol moiety. is the part of
A is an antibody or antigen-binding fragment thereof optionally modified to have a ligase donor substrate recognition motif or a ligase acceptor substrate recognition motif, e.g., A is a human epidermal growth factor receptor 2 (HER2) an antibody that binds, for example trastuzumab, which is modified to have a ligase donor substrate recognition motif or a ligase acceptor substrate recognition motif;
The antibody drug conjugate composition according to <52>, <53> or <54>, wherein z is an integer of 1 or 2.

<56> A is trastuzumab with a C-terminally modified light chain (LCCT _L ) modified by introduction of a short peptide spacer such as -GA- and a ligase donor substrate recognition motif LPXTGJ, where J in LPXTGJ is The antibody-drug conjugate composition according to <55>, which is absent or is an amino acid fragment containing 1 to 10 amino acids.

<57> The payload is DM1, L ³ does not exist, and formulas (XIX-1-1) to (XX-1-1) have structures (vii) and (viii), respectively, <55> or <56>.

In the tenth aspect, the treatment methods provided in the present disclosure can be described as <58> to <60> below.

<58> A method for treating cancer in a patient in need of cancer treatment, the method comprising administering to the patient an effective amount of the antibody drug conjugate composition according to any one of <53> to <57>. The method described above, comprising:

<59> The method according to <58>, wherein the cancer is a HER2-positive cancer, such as a HER2-positive breast cancer.

<60> The method according to <58> or <59>, wherein the antibody drug conjugate composition is as described in <57>.

Pharmaceutical Compositions, Formulations, and Kits In another aspect, the present disclosure further provides pharmaceutical compositions comprising (a) and (b):
(a) a composition comprising a compound of formula (XI), (XII), (XIII) or (XIV), or an antibody drug conjugate of the invention, or a mixture of compounds of formula (XIII) and (XIV); or Antibody drug conjugate compositions of the present disclosure.
(b) A pharmaceutically acceptable carrier.

Treatment and Uses In another aspect, the present disclosure provides a compound of formula (XI), (XII), (XIII) or (XIV), or an antibody drug conjugate of the invention, or a compound of formula (XIII) and (XIV). Further provided is the use of a composition comprising a mixture of compounds, or an antibody drug conjugate composition of the present disclosure, or a drug composition of the present disclosure, in the preparation of a medicament for treating cancer.

In another aspect, the present disclosure provides a compound of formula (XI), (XII), (XIII) or (XIV), or an antibody drug conjugate of the invention, or a mixture of compounds of formula (XIII) and (XIV). Further provided is a method of treating cancer comprising administering to a subject in need thereof an effective amount of a composition comprising: or an antibody drug conjugate composition of the present disclosure, or a drug composition of the present disclosure.

In a further aspect, the invention provides a compound of formula (XI), (XII), (XIII) or (XIV), or an antibody-drug conjugate of the invention, or a compound of formula (XIII) and ( XIV), or antibody drug conjugate compositions of the present invention, or drug compositions of the present disclosure.

In one embodiment, the cancer is a HER-2 positive cancer.

Beneficial Effects The invention of the present disclosure achieves at least one of the following technical effects (1) to (5).
(1) Complete separation and purification of the four isomeric compounds contained in mixture 1 can be achieved, and pure isomeric compounds can be obtained with high efficiency and yield.
In addition, in the separation process of β1, β2, and α isomers (for example, the process of Example 1) and the analysis process (for example, the process of Example 2), the acidic agent used in the mobile phase is TFA, NaH ₂ PO ₄ , CH _2O2 , _K2HPO4 , AA, TEAP, _NaClO4 or other conventional reagents, little separation _between the β1, β2 _and α isomers was observed, whereas _CH3 as the acidic agent It should be noted that when choosing COOH and L-TA, one can successfully separate and purify the β1, β2 and α isomers. Therefore, the inventor believes that a particular acidic agent, _CH3COOH or similar alkyl carboxylic acid, can effectively increase the degree of separation between β1, β2 and α isomers, thereby improving the separation and/or purification method. I unexpectedly discovered that it can be applied to
(2) The method is easy to operate, has low cost, and has been proven effective in pilot scale production, making it advantageous for industrialized production.
(3) Separation effectiveness of complex samples is usually monitored by complex and expensive after-column detection, e.g. LCMS methods use expensive chromatography columns to accurately analyze and in some cases It is necessary to pre-treat the sample to remove any remaining non-volatile salts in the product obtained from the separation.
The analytical method of the present disclosure uses similar chromatographic conditions as the separation method and therefore requires less adjustment when designing the chromatographic method of the separation process or the chromatographic method of the analytical process, and the chromatographic processing of complex samples. becomes easier. Additionally, since volatile reagents are used in the separation method of the present disclosure, the product obtained by separation does not contain nonvolatile salts and can be directly subjected to LCMS analysis without pretreatment for desalting. This is particularly advantageous if mixture 1 is the reaction mixture of a ring-opening reaction. When necessary, impurities or by-products can be detected quickly.
(4) The method has shown high effectiveness, e.g. high resolution (parameter R in chromatography) and sufficient precision.
(5) Separated isomers may exhibit differences in cytotoxicity. In the human breast cancer HCC1954 cell proliferation assay, isomer α2 is twice as active as the β isomer (mixture of β1 and β2 isomers).

The α-ADC of the present disclosure achieves at least one of the following technical effects.
α-ADCs have significant antitumor effects and can be concentrated in tumors and maintain stability in situ. Compared to Kadcyla, α-ADC showed similar effects at lower doses, demonstrating the superior efficacy of α-ADC of the present disclosure.

EXAMPLES In order to more clearly explain the objectives and technical solutions of the present disclosure, the present disclosure will be further explained below with specific examples. It should be understood that these examples are not intended to limit the scope of the invention. All specific experimental methods not described in the following examples are performed according to conventional experimental methods.

abriviation

Equipment, Materials, and Reagents Unless otherwise specified, equipment and reagents may be commercially available or prepared by methods conventional in the art. Reagents can be used directly without further purification.

¹ H NMR, ¹³ C NMR, ¹ H- ¹ H COSY and HMBC spectra are recorded on a Bruker AV-600, 600 MHz. Chemical shifts are expressed in parts per million (ppm). Coupling constants are in Hertz (Hz). Splitting patterns describe the apparent multiplicity and include s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad line). ) is specified.

Cell viability is tested using the CellTiter-Glo ^® Luminescent Kit (Promega, Cat. #G7573) read on a Biotek Cytation 3 imaging reader.

The HCC1954 human breast cancer xenograft model (HCC1954 model) and the NCI-N87 human gastric cancer xenograft model (NCI-N87 model) in female BALB/c nude mice are provided by ATCC. The cynomolgus monkey is from GuangZhou XiangGuan, Ltd. provided by.

PLT, APTT, AST, ALT, GLOB and A/G are tested on a hematology analyzer (ADVIA 2120i Siemens) and auxiliary reagents.

Kadcyla is provided by Roche.

Preparation of mixture containing β isomer and α isomer A compound having the following structure is prepared, and a ring-opening reaction of the thiosuccinimide group is performed in the same manner as the method described in WO2015165413A1.

The ring-opening reaction yields four isomers (isomeric compounds (i-1), (ii-1), (iii-1) and (iv-1)). The difference in that the thiol group is located in the alpha or beta position of the uncleaved amide bond is why they are named alpha and beta isomers, respectively. The structures of the four isomers are as follows.

The reaction mixture obtained from the reaction is directly subjected to the separation process described below, said reaction mixture containing said four isomers.

Example 1 Separation of β1, β2 and α isomers 1.1 Separation process Gradient elution is used for the separation of β1, β2 and α isomers. The elution gradient includes a pH gradient and an organic solvent gradient. The chromatography conditions are as shown in Table 1.

式中、＊は不斉中心（２２－Ｃ）を表す。 In the figure, peak 1 (first elution peak within the range of about t _R 27 min to about t _R 39 min), peak 2 (second elution peak within the range of about t _R 27 min to about t _R 39 min), and peak 3 (t _R Three main peaks are shown: peaks within the range 42 min to t _R 51 min). The purity of each fraction of the eluent is detected by HPLC using the analytical method described in Example 2. Fractions are combined based on the analysis results and then lyophilized for NMR assay. According to the 1D and 2D NMR data, peak 1 and peak 2 each contain two β isomers. The structure of the β isomer is as follows.

In the formula, * represents an asymmetric center (22-C).

The β isomer contained in peak 1 is designated as β1. ¹ H NMR, ¹³ C NMR, ¹ H- ¹ H COSY and HMBC data are shown in Table 2.

The β isomer contained in peak 2 is designated as β2.

¹ H NMR, ¹³ C NMR, ¹ H- ¹ H COSY and HMBC data are shown in Table 3.

According to the 1D and 2D NMR data, peak 3 contains the α isomer. The content of peak 3 is estimated from the mechanism of the ring-opening reaction, and the peak is expressed as a mixture of two α isomers, ie, a mixture of α1 and α2.

Separations under other chromatographic conditions were tested and the results are shown in sections 1.2 to 1.6 below.

1.2 Stationary Phase Packed columns from different manufacturers are tested. The chromatography conditions are as shown in Table 4.

The HPLC diagrams are as shown in FIGS. 1 to 4.

The purity of each fraction of the eluent is detected by HPLC using the analytical method described in Example 2. In the figure, three main peaks are shown. Fractions are combined based on the analysis results and then lyophilized for NMR assay. According to the analytical results and 1D and 2D NMR data, under the above chromatographic conditions, the elution order of the isomers is the same as in Example 1. In Figures 2-4, the two β isomers each elute in the first two peaks, and the mixture of the two α isomers elutes in the third peak.

The factors were calculated and shown in Table 5.

R greater than 1 was observed for all types of RP-C18 stationary phases. The disclosed method is robust as it is applicable to a variety of commercially available packed columns.

1.3 Acid as acidic agent 1

Various acids were tested as Acidic Agent 1 in Eluent A. The chromatography conditions are as shown in Table 6.

The HPLC diagrams of the separations are shown in Figures 5-13.

According to FIGS. 5, 7, 11 and 12, moderate resolution is observed when optionally substituted alkyl carboxylic acids are used, among which AcOH and L-TA show good resolution. Inorganic acids (eg phosphoric acid) also show a certain degree of resolution. Formic acid has a low resolution. The separation ability of a mobile phase can be affected by the acid groups of organic acids.

The purity of each fraction of the eluent is detected by HPLC using the analytical method described in Example 2. Fractions are combined based on the analysis results and then lyophilized for NMR assay. According to the analytical results and 1D and 2D NMR data, under the above chromatographic conditions, the elution order of the isomers is the same as in Example 1. In Figures 11-13, the two β isomers are eluted in the first two peaks (which partially fuse with each other), and the mixture of the two α isomers is eluted in the third peak.

1.4 Buffer Salt as Acidic Agent 1 Phosphate buffer was tested as acidic agent 1 in Eluent A.

The test results for eluent A are shown in Table 7, and the other chromatography conditions other than eluent A are the same as in Table 6.

The HPLC diagrams of the separations are shown in Figures 14-19.

Summarizing the results of 1.3 and 1.4, it can be seen that pH value is not the only factor that influences solute chromatography behavior. Separation of isomers is greatly influenced by the acid agent material used. Applicants have unexpectedly discovered that among the acidic agents tested, AcOH and L-TA provide significantly increased resolution compared to other acidic agents.

1.5 Organic Solvent The degree of separation between the two α isomers was analyzed by comparison with Example 3, and a higher degree of separation was obtained when MeOH was used as the organic solvent. It has been shown that this may be an important factor.

1.6 Gradient Different gradients are used in 1.1, 1.3 and 1.4 above, and isocratic elution is used in 1.2 above, allowing the method of the present disclosure to be applied to a range of elution compositions. Shows that it can be tolerated. This provides flexibility to the method, which can be particularly beneficial in pilot scale manufacturing, and contributes to relieving pressure on robust control of elution compositions.

Further tests are performed using 0.3% aqueous CH ₃ COOH as eluent A and the elution gradients in Tables 8-1 and 8-2. Chromatography conditions other than eluent A and elution gradient are the same as in Table 6.

The HPLC diagram of the separation is as shown in Figures 20-21.

The purity of each fraction of eluent is detected by HPLC using the analytical method described in Example 2. Fractions are combined based on the analysis results and then lyophilized for NMR assay. According to the analytical results and 1D and 2D NMR data, under the above chromatographic conditions, the elution order of the isomers is the same as in Example 1. In Figures 20-21, the two β isomers are eluted in the first two peaks (which partially fuse with each other) and the mixture of the two α isomers is eluted in the third peak.

The factors were calculated and shown in Table 9.

A B gradient of 46% to 56% within 80 min provides higher resolution than a B gradient of 46% to 66% within 30 min. Given the improved resolution, the tailing factor (improved) and theoretical plate number (reduced) for slope 2 are still acceptable.

Further extension of the elution time beyond 80 min has shown unsatisfactory results in system applicability testing.

Example 2 Process control of β1, β2 and α isomer separation In Example 2, the method described in Example 1 was applied on an analytical scale to obtain a particle size smaller than that used in Example 1 (10 um). ) is used. The chromatography conditions are as shown in Table 10.

The HPLC diagram of the separation is as shown in FIG.

By calculation, the degree of separation is 1.28 and the theoretical plate number is 20020.6.

式中、＊は不斉中心（２２－Ｃ）を表す。 Example 3 Separation of α1 and α2 isomers 3.1 Separation method The α isomer obtained in Example 1 is further separated to obtain isomer α1 and isomer α2. The structure of the α isomer is as follows.

In the formula, * represents an asymmetric center (22-C).

Gradient elution is used to separate the α1 and α2 isomers from the α isomer obtained in Example 1. The chromatography conditions are as shown in Table 11.

The HPLC diagram of the separation is as shown in FIG. In the figure, two main peaks are shown: peak 1' (t _R 40.075 min) and peak 2' (t _R 42.497 min). The purity of each fraction of eluent is detected by HPLC using the analytical method described in Example 4. The α isomer contained in peak 1 is designated as α1. ¹ H NMR, ¹³ C NMR, ¹ H- ¹ H COSY and HMBC data are shown in Table 12.

The α isomer contained in peak 2 is designated α2. ¹ H NMR, ¹³ C NMR, ¹ H- ¹ H COSY and HMBC data are shown in Table 13.

Separation under other chromatographic conditions was tested and the results are shown in sections 3.2 to 3.6 below.

3.2 Acid as Acidic Agent 3 Various acids were tested as acidic agent 3 in Eluent C. The chromatography conditions are as shown in Table 14.

The HPLC diagram of the separation is shown in Figures 24-31.

3.3 Buffer Salt as Acidic Agent 3 Phosphate buffer was tested as acidic agent 1 in Eluent A.

The test results for Eluent C are shown in Table 15, and the other chromatography conditions other than Eluent C are the same as Table 14.

The HPLC diagrams of the separations are shown in Figures 32-44.

3.4 Summary of Acidic Agent 3 The factors were calculated and shown in Table 16.

As acidic agent 3, 0.1% TFA, 10mM _K2HPO4 ( _PH =2.0) _, 10mM _K2HPO4 (PH=4.0), 10mM TEAP (PH=2.0), or 10mM When using TEAP PH=4.0, a resolution greater than 1 is achieved.

Compared to the method of Example 1, in Figures 23-44 and Table 16, the substance of the acidic agent shows less significant influence, demonstrating the difference in chromatographic properties of the β and α isomers.

Example 4 Process Control of the Separation of α1 and α2 Isomers In Example 4, the method described in Example 3 was applied on an analytical scale to obtain a particle size smaller than that used in Example 3 (10 um). 5 um) is used. The chromatography conditions are as shown in Table 17.

The HPLC diagram of the analysis is shown in Figure 45.

The analytical methods of the present disclosure can be performed using the parameter sets shown in any one of Tables 17-19. The flexibility of the method lends itself to its application in different conditions and can also meet the detection requirements of different stages in the target product preparation process, such as for example the preparation of bioconjugates.

Example 5 Cytotoxicity of β-isomer, α1-isomer and α2-isomer The peaks eluted in Example 1 were collected, where: (1) the first peak and the second peak were collected as a sample (β-isomer); and (2) collect the third peak as a sample (α isomer) and separate it using the method described in Example 3 to obtain the α1 and α2 isomers.

Cytotoxicity of the β, α1 and α2 isomers is tested by tumor cell proliferation assay.

The detection method is as follows.

Human breast cancer HCC1954 cells in good growth conditions with a degree of cell confluence of approximately 80% are digested with 0.25% trypsin, harvested, and placed in 96-well plates for overnight culture. After cell attachment, the test substance is diluted 3-fold from 10 nM in a total of 10 gradients. After 72 h of culture at 37° C. in an incubator, cell viability is detected with the CellTiter-Glo® luminescence kit. The formula for cell viability is Viability = (RLU(X)-RLU(Puro))/(RLU(Control)-RLU(Puro))*100%. Data processing, IC50 calculation and curve fitting are performed using a Logistic model (4 parameters) by Prism 6 software. The results are shown in FIG. 46 and Table 20.

As shown in Table 20, the activity of the α1 isomer is within ±30% of the β isomer activity, so their activities are considered equivalent. The relative activity of the α2 isomer is 205% (the biological activity of the β isomer is set as 100%), ie the activity of the α2 isomer is about twice that of the β isomer.

Example 6 Preparation of α-ADC The pure product containing the α isomer (mixture of α1 and α2) obtained in Example 1 is subjected to a conjugation reaction with a therapeutic antibody. The conjugation reaction method is similar to the method described in WO2015165413A1. The resulting product is a mixture of ADCs and is designated as "α-ADC". α-ADC prepared by conjugating α-isomer with T-LCCT _L -HC (T represents trastuzumab) as described in WO2015165413A1 is designated as “α-ADC Composition 1” .

Example 7 HER2-selective cytotoxicity of HER2 targeting α-ADC Purpose: To evaluate the selective cytotoxicity of α-ADC, HER2-positive cells HCC1954 and HER2-negative cells MDA-MB-468 were incubated with α-ADC, respectively. - Treatment with ADC Composition 1, Kadcyla and DM1.

Research design:

The research design is as shown in Tables 21 and 22. After incubating cells with α-ADC composition 1, Kadcyla or DM1 for 72 h, cell viability is detected with the CellTiter-Glo® luminescence kit. The formula for cell viability is Viability = (RLU(X)-RLU(Puro))/(RLU(Control)-RLU(Puro))*100%. Data processing, IC50 calculation and curve fitting are performed using a Logistic model (4 parameters) by Prism 6 software.

Results: The results are shown in FIG. 47 and Table 23.

α-ADC showed no inhibitory effect on HER2-negative cells up to 100 nM, but showed a significant inhibitory effect on HER2-positive cells. T-DM1, but not α-ADC, exhibited off-target cytotoxicity in HER2-negative cells at high concentrations. Such off-target cytotoxicity may be due to DM1 reduction due to reverse Michael reaction of MCC-DM1 in T-DM1. The MCC' linker (containing an open ring MCC structure) of α-ADC is designed to reduce the reverse Michael reaction, thereby avoiding or significantly reducing the shedding of DM1 before internalization. Ru. This indicates that α-ADC is safer than T-DM1 in normal cells.

Example 8 Biodistribution properties of α-ADC Purpose: ⁸⁹ Zr-α-ADC Composition 1 was analyzed by positron emission computed tomography/computed tomography (PET/CT) scans at different time points after a single intravenous injection. , to study the biodistribution properties of ⁸⁹ Zr-α-ADC in tumor-bearing mice.

Study design: [ ^89Zr ] isotope labeling method was used to detect α in BT-474 human breast cancer xenografts in athymic mice (referred to as BT-474 xenograft tumor model) after a single intravenous injection. - Study the distribution of ADC composition 1. The passed ⁸⁹ Zr-α-ADC Composition 1 is administered to experimental animals. ⁸⁹ Zr-α-ADC Composition 1 is administered to eight BT-474 xenograft tumor models. PET/CT scans are performed 1 h, 24 h, 48 h, 96 h, 168 h, and 336 h after administration, and static scans are performed for 10-30 min.

Results: The results are shown in FIG.

After a single intravenous injection of ⁸⁹ Zr-α-ADC Composition 1 into the BT-474 xenograft tumor model, the total radioactivity was mainly distributed within the tumor, followed by blood-rich organs (liver, heart). , kidneys, spleen, lungs) and knees, and to a lesser extent in other tissues and organs (bones, brain, muscles). Calculated based on radioactivity, the AUC (0-336h) order of ⁸⁹ Zr-α-ADC Composition 1 in different tissues is: tumor>liver>knee>heart>kidney>spleen>lung>bone>muscle>brain. be. These results indicate that ⁸⁹ Zr-α-ADC Composition 1 hardly passes through the blood-brain barrier and that the tumor target of ⁸⁹ Zr-α-ADC Composition 1 is clear.

After a single intravenous injection of ⁸⁹ Zr-α-ADC composition 1 into the BT-474 xenograft tumor model, the ratios (tumor to muscle and tumor to heart) gradually increased, both at 336 hours after administration. reached the highest at 34.78 and 15.54, respectively.

Example 9 In Vivo Anti-Tumor Efficacy of α-ADC Purpose: The purpose of this study was to test subcutaneous HCC1954 human breast cancer xenograft model (HCC1954 model) and NCI- To evaluate the in vivo antitumor effect of α-ADC in the N87 human gastric cancer xenograft model (NCI-N87 model).

Research design: The research design is as shown in Tables 24 and 25.

The therapeutic effect is calculated as follows. T/C (%) = (Mean RTV of treatment group)/(Mean RTV of control group) x 100%. TGI = [1 - (mean tumor volume at the end of treatment for the treatment group - mean tumor volume at the start of treatment for the treatment group) / (mean tumor volume at the end of control group treatment - mean tumor at the start of treatment for the control group) volume)]×100%.

Results: The therapeutic efficacy of α-ADC Composition 1 (DAR 1.79) administered alone is evaluated and compared to the positive control Kadcyla (DAR ~3.5) in the HCC1954 human breast cancer xenograft model. The results of tumor size of different groups at different time points after the start of treatment are shown in Figure 49. Compared to the vehicle group, test article α-ADC composition 1 at 1.25 mg/kg, 2.5 mg/kg and 5 mg/kg (T/C was 18.83%, 9.12% and 2.12%, respectively) and TGI are 86.61%, 96.97% and 104.43%, p are 0.002, 0.001 and 0.001, respectively), and 2.5mg/kg and 5mg/ kg of Kadcyla (T/C are 6.47% and 1.57%, respectively, TGI is 99.79% and 105.02%, respectively, p is 0.001 and 0.001, respectively) Treatment resulted in significant antitumor activity. Their average tumor sizes are 418, 202, 47, 144 and 35 mm, ^respectively , at the same time. α-ADC Composition 1 and Kadcyla at the same dose level produced similar antitumor activity in the HCC1954 xenograft model, with p-values of 0.791 (5 mg/kg) and 0.701 (2.5 mg/kg). ).

The therapeutic efficacy of α-ADC composition 1 administered alone in the NCI-N87 human gastric cancer xenograft model is evaluated. The results of tumor size of different groups at different time points after tumor inoculation are shown in Figure 49. Compared to the vehicle group, test article α-ADC Composition 1 at 5 mg/kg and 15 mg/kg had T/C of 34.13% and 5.07%, respectively, and TGI of 76.62% and 110. 38%, p is 0.010, 0.001, respectively), and 5 mg/kg, 15 mg/kg Kadcyla (T/C is 41.61% and 4.66%, respectively, TGI is 67.93% and 110.87%, p = 0.022, 0.001, respectively) resulted in significant antitumor activity. Their average tumor sizes are 475, 71, 579 and 65 mm, ^respectively , at the same time. On the 35th day after treatment, the tumor volume when 1.67 mg/kg of α-ADC composition 1 was administered was 1025 mm ⁽ T/C = 73.77%, TGI = 30.67%, p = 0 .509). α-ADC Composition 1 and Kadcyla at the same dose level produced similar anti-tumor activity with p-values of 0.791 (5 mg/kg) and 1.000 (15 mg/kg), respectively.

Example 10 Pharmacokinetic properties of α-ADC Purpose: The purpose of this study is to evaluate the pharmacokinetic properties of α-ADC after a single administration in cynomolgus monkeys.

Study design: 0 mg/kg (20 min/each administration/monkey) of vehicle (α-ADC composition 1 preparation buffer) or 10, 30 and 45 mg/kg of α-ADC composition 1 in a total of 24 cynomolgus monkeys. A single intravenous injection is given to (3 animals/sex/group, 4 groups total). Animals will be necropsied on day 43 after a 6 week observation period. Details of the number of animals and dose levels are shown in Table 26.

Blood sampling time points include 0h, 5min, 1h, 4h, 8h, 24h, 48h, 72h, 168h, 336h, 504h, 672h, 840h and 1008h.

Results: The combined pharmacokinetic data is shown in Figure 50.

After IV infusion, serum concentrations of α-ADC Composition 1 and α-ADC Composition 1 TAb reach peak values substantially rapidly and decrease in an almost biphasic manner, with α-ADC Composition 1 group having α- ADC Composition 1 decreased faster than TAb. The exposure (average C _max , AUC _0-t and AUC _0-∞ ) of α-ADC Composition 1 and TAb increases approximately dose-proportionally. Little or no prolongation of mean MRT was observed with increasing doses of α-ADC Composition 1. No obvious sex differences were observed in C _max , AUC _0-t or AUC _0-∞ for α-ADC Composition 1, α-ADC Composition 1 TAb, and α-ADC Composition 1 mAb. The T _1/2 of α-ADC Composition 1 is 3.4-4.1 days, and the TAb T _1/2 of α-ADC Composition 1 is 5.2-8.6 days.

At 5 min post-dose, DM1 slightly higher than LLOQ was detected in the group with α-ADC composition 1 at ≧30 mg/kg, and at 1 h after administration, in the group with α-ADC composition 1 at 45 mg/kg. Only DM1 was detected slightly higher than LLOQ. The mean C _max and AUC _0-t of DM1 increase as the dose increases from 30 to 45 mg/kg.

Example 11 Toxicity and Potential Target Organs of α-ADC Objectives: The research objectives include i) and ii) below. i) Assess toxicity and potential target organs after repeated intravenous infusions (once every 3 weeks, 3 times in total) of α-ADC in cynomolgus monkeys during a main phase of 7 weeks and after a recovery period of 6 weeks. Assess the reversibility of observed or potential delayed toxicity to support the study design of subsequent toxicity studies and clinical trials. ii) Characterize the toxicokinetics of α-ADC in cynomolgus monkeys.

Study design: 40 cynomolgus monkeys (5 animals/sex/group, total 4 groups) were randomly assigned to the study to receive 3 doses of α-ADC composition 1 (10, 30 and 45 mg/kg) or vehicle (0 mg/kg). It is administered once a week, for a total of 3 doses over a 7-week dosing phase. At the end of the dosing phase, 3 animals/sex/group will be necropsied, and the remaining 2 animals/sex/group will be necropsied after a 6 week recovery period. The results are shown in Table 27.

Results: The combined toxicokinetic data is shown in Figure 51.

After IV infusion, serum concentrations of α-ADC Composition 1 (10, 30 or 45 mg/kg), α-ADC Composition 1 and TAb typically reach peak values quickly and decrease in an approximately biphasic manner, respectively. Thereafter, it decreased over time, with α-ADC Composition 1 decreasing faster than TAb on both days 1 and 43. The average serum concentration of α-ADC Composition 1 is close to, but slightly lower than, that of TAb. The exposure (average C _max and AUC _0-t ) of α-ADC Composition 1 and TAb increases approximately dose-proportionally. No gender differences were observed regarding the exposure amount for α-ADC Composition 1 and TAb. Based on the day 1 exposure (AUC _0-t ), the average accumulation rate at day 43 for both α-ADC Composition 1 and TAb was approximately 1.0 for all doses, with no apparent No drug accumulation was shown.

On days 1 and 43, the highest post-dose DM1 was detected at three time points (5 min, 1 h and 4 h) post-dose only in the 30 and 45 mg/kg α-ADC Composition 1 treatment groups. , slightly higher than LLOQ. The mean C _max and AUC _0-t of DM1 increase as the dose increases from 30 to 45 mg/kg.

The highest non-severe toxicity dose (HNSTD) is determined to be 45 mg/kg.

The notable findings are as follows.

At 45 mg/kg: decrease in PLT (males), prolongation of APTT (males), increase in AST and/or ALT are observed regularly on day 7 post-dose of each dosing cycle, with recovery thereafter. GLOB decreases. A decrease in A/G was observed during the administration phase. Increased spleen weight (absolute and relative) was also present in both genders. Histopathological changes included enlarged Kupffer cells in the liver, increased hypercellularity and single-cell necrosis in the red pulp of the spleen, epithelial cell necrosis in the cornea (one male) and duodenal Brunner's gland, and Includes increased mitotic figures of Kupffer cells in the liver, splenic histiocytes, duodenal Brunner's gland epithelium (female), corneal epithelium (one male), and esophageal squamous epithelium (male). Chronic perivascular inflammation and fibrosis was present at the injection site in one female. Although all the above changes were not observed at the end of the recovery period, in one male an increase in the mesangial matrix of bilateral glomeruli was observed, as well as ADA was detected from day 43, and therefore changes may be due to the immune system. It is thought to be related to the origin of the disease.

In summary, under the current study conditions, repeated intravenous infusions (once every 3 weeks, 3 times in total) with α-ADC Composition 1 at 10, 30 and 45 mg/kg were tolerated by cynomolgus monkeys, and a single administration The amount can be up to 45 mg/kg. Potential target organs/tissues are liver, spleen, epithelium (duodenum, esophagus and cornea) and site of administration.

Compared to Kadcyla, α-ADC Composition 1 releases much less DM1 into the circulation and may therefore have fewer side effects. A detailed comparison is shown in Table 28. The DM1 C _max of α-ADC Composition 1 is approximately 1% of T-DM1 at a dose of 30 mg/kg, and the DM1 AUC _0-t of α-ADC Composition 1 is approximately 1% of T-DM1 at a dose of 30 mg/kg. It is approximately 0.002%.

Typical pathological evaluation items for α-ADC and Kadcyla are shown in Table 29.

Pathological evaluation shows that the main DM1-related toxicities are hepatotoxicity and peripheral neuropathy. Compared to Kadcyla at 10 mg/kg, the HNSTD dose of α-ADC Composition 1 (45 mg/kg) shows significantly less severity in histopathology. Since α-ADC Composition 1 and Kadcyla at the same dose level produce similar antitumor activity and the HNSTD of α-ADC Composition 1 is 4.5 times that of Kadcyla, the therapeutic window of α-ADC Composition 1 is It should be much wider than Kadcyla.

It should be understood by those skilled in the art that various changes and modifications can be made to the present disclosure without departing from the spirit and scope of the disclosure. The embodiments described herein are provided by way of example only and should not be construed as limitations. The true scope and spirit of the invention is limited by the claims, and the specification and examples are considered to be exemplary only.

Claims (60)

A compound of formula (XI), (XII), (XIII) or (XIV),

During the ceremony,
The payload is a cytotoxin moiety containing one or more chiral centers;
L ¹ is absent or C _1-10 alkylene group, C _3-10 cycloalkylene group, C _6-10 arylene group, _4-10 membered heterocyclylene group, 5-10 membered heteroarylene group, -NH- , -(CO)-, -NH(CO)-, and -(CO)NH-,
L ² is a bond, or C _1-10 alkylene group, C _3-10 cycloalkylene group, C _6-10 arylene group, _4-10 membered heterocyclylene group, 5-10 membered heteroarylene group, -NH one or more divalent groups selected from the group consisting of -, -(CO)-, -NH(CO)-, and -(CO)NH-,
M contains a ligase recognition motif;
The compound may be primarily in pure or substantially pure isomeric form, e.g. substantially free of other isomeric forms, e.g. greater than 90%, e.g. greater than 95%, e.g. greater than 98%, e.g. Said compound having a purity of at least 99%. 2. A compound according to claim 1, which is a compound of formula (XIII) or (XIV). The ligase recognition motif is
(a) LPXTG, for example, where X is any amino acid residue, such as LPETG;
(b) The compound according to claim 1 or 2, wherein G is glycine (Gly) and _n is an integer from 3 to 10. 4. A compound according to claim 3, wherein M is H-Gly-Gly-Gly-Lys-NH ₂ and M is linked to L ² via the ε-amino group on the lysine. L ¹ does not exist,
L ² is

A compound according to any one of claims 1 to 4, selected from: L ¹ does not exist,
L ² is

and
M is

and
During the ceremony,
Y is a bond or a spacer sequence of 1 to 20 amino acids;
n is an integer from 3 to 10, for example 3;
x is selected from the group consisting of hydrogen, -OH, _-NH2 , amino acid fragments containing 1 to 10 amino acids, and nucleotide fragments containing 1 to 10 nucleotides, such as -OH, _-NH2 , and A compound according to claim 1, 2, 3 or 4 selected from amino acid fragments comprising 1 to 10 amino acids, for example selected from the group consisting of -OH, _-NH2 and Gly, for example _-NH2 . . 7. A compound according to any one of the preceding claims, wherein the cytotoxin comprises a thiol moiety capable of reacting with a maleimide moiety. the cytotoxin is a maytansinoid;
For example, DM1

The compound according to any one of claims 1 to 7, which is A compound according to any one of the preceding claims, selected from compounds of formula (i), (ii), (iii) or (iv).

10. A compound according to claim 9, which is a compound of formula (iii) or (iv). An antibody drug conjugate comprising an open ring thiosuccinimide structure of formula (I), (II), (III) or (IV),

The compound may be primarily in pure or substantially pure isomeric form, e.g. substantially free of other isomeric forms, e.g. greater than 90%, e.g. greater than 95%, e.g. greater than 98%, e.g. An antibody drug conjugate having a purity of at least 99%. 12. The antibody-drug conjugate of claim 11, formed by reacting the compound of any one of claims 1 to 11 with an antibody in the presence of a ligase. Each has formulas (XVII-1-1) to (XX-1-1),

During the ceremony,
The payload is as defined above, e.g. a cytotoxin moiety containing one or more chiral centers, e.g. the remainder of the maytansinoid molecule excluding the thiol moiety, e.g. the remainder of DM1 without the thiol moiety. is the part of
A is an antibody or antigen-binding fragment thereof optionally modified to have a ligase donor substrate recognition motif or a ligase acceptor substrate recognition motif, e.g., A is a human epidermal growth factor receptor 2 (HER2) an antibody that binds, for example trastuzumab, which is modified to have a ligase donor substrate recognition motif or a ligase acceptor substrate recognition motif;
13. The antibody drug conjugate according to claim 11 or 12, wherein z is an integer of 1 or 2. A is trastuzumab with a C-terminally modified light chain (LCCT _L ) modified by the introduction of a short peptide spacer such as -GA- and a ligase donor substrate recognition motif LPXTGJ, where J in LPXTGJ is absent or , or an amino acid fragment comprising 1 to 10 amino acids. The payload is DM1,
L ³ does not exist,
The antibody-drug conjugate according to claim 13 or 14, wherein formulas (XVII-1-1) to (XX-1-1) have structures (v) to (viii), respectively.

The antibody drug conjugate according to claim 14, having the structure (vii) shown in claim 15. A method for preparing a compound according to any one of claims 1 to 10, comprising:
(i) synthesizing a mixture of compounds of formulas (XI), (XII), (XIII) and (XIV);
(ii) subjecting the mixture to chromatography to obtain a target compound,
said step in which each of said target compounds is obtained as a separate product or the two target compounds are obtained as a second mixture;
(iii) optionally collecting an eluate comprising a third mixture collected in step (ii), said third mixture containing one or more additional target compounds different from the target compound separated in step (2); chromatography of the collected eluate containing a target compound to separate said additional target compound. 18. The method of claim 17, wherein the chromatography is reverse phase chromatography. 17. A method of treating cancer in a patient in need of cancer treatment, said method comprising administering to said patient an effective amount of an antibody drug conjugate according to any one of claims 11 to 16. . 20. The method of claim 19, wherein the cancer is HER2-positive breast cancer and the antibody drug conjugate is as described in claim 16. A method of separating one or more target compounds from a mixture 1, wherein the mixture 1 comprises four compounds, each of the four compounds comprising part 1, part 2 and part 3;
Part 1 has an open ring thiosuccinimide structure selected from formulas (I) to (IV),

here,
The thiol group and the amide group in moiety 1 constitute two linking sites, moiety 2 is linked to moiety 1 through one of them, moiety 3 is linked to moiety 1 through the other,
Portion 2 contains one or more chiral centers;
Moiety 1 in the four compounds is different,
Part 3 is the remaining part in the molecule,
The molecular weight of portion 3 is less than or equal to 1900;
the one or more target compounds are selected from the four compounds contained in mixture 1;
The method includes:
(1) preparing mixture 1;
(2) chromatography of mixture 1 to obtain the target compound;
here,
a. each of said target compounds is obtained as a separate product, or b. Said method, wherein the two target compounds are obtained as a mixture, said mixture being defined as mixture 2. (3) collecting an eluate containing mixture 3 collected in step (2), wherein mixture 3 contains one or more additional targets different from the target compounds separated in step (2); said step comprising a compound;
22. The method of claim 21, further comprising: (4) subjecting the eluate collected in step (3) to chromatography to separate the additional target compound. In step (2) and step (4), four target compounds are obtained, two of which are obtained as separate products in step (2) and the other two target compounds are obtained in step (4). obtained as a separate product,
Preferably, moiety 1 in the two target compounds obtained as separate products in step (2) each has the structure of formula (I) or formula (II), and in step (4) the moieties 1 in the two target compounds obtained as separate products 23. The method according to claim 21 or 22, wherein moiety 1 in the two target compounds obtained as 1 has a structure of formula (III) or formula (IV), respectively. In step (2) four target compounds are obtained, two of which are obtained as separate products and the remaining two target compounds are obtained as mixture 2;
Preferably, moiety 1 in the two target compounds obtained as separate products in step (2) each has the structure of formula (I) or formula (II), and the two targets obtained as mixture 2 22. The method according to claim 21, wherein moiety 1 in the compound has a structure of formula (III) or formula (IV), respectively. The chromatography in step (2) is reversed phase chromatography, and/or the stationary phase used in the reversed phase chromatography in step (2) is selected from alkyl-bonded silica gel species, and/or The mobile phases in are eluent A and eluent B,
Eluent A is water optionally containing acidic agent 1;
Eluent B is an organic solvent 1 optionally containing an acidic agent 2;
However, the condition is that at least one of acidic agent 1 and acidic agent 2 is present,
For the separation range, B is a slope from about 0% to 30% to about 30% to 100%, with the remainder being A.
25. A method according to any one of claims 21 to 24, wherein the acidic agent 1 and acidic agent 2 are independently inorganic or organic acids. Acidic agent 1 is selected from AcOH and L-tartaric acid (L-TA), preferably acidic agent 1 is AcOH, and/or the amount of acidic agent 1 is about 0 based on the total volume of eluent A. .01% to about 1%, preferably about 0.05% to about 0.5%, more preferably about 0.1% to about 0.5%, most preferably about 0.1% to about 0.3%. %, in particular 0.3%. Acidic agent 2 is not present, or the type of acidic agent 2 is the same as acidic agent 1,
and/or The amount of acidic agent 2 is about 0.01% to about 1%, preferably 0.05% to about 0.5%, more preferably about 0.1% based on the total volume of eluent B. to about 0.5%, most preferably about 0.1% to about 0.3%, especially 0.3%, and/or Acidic Agent 1 and Acidic Agent 2 (if present) in the elution composition. 27. The method of claim 26, wherein the total amount of case) is about 0.005-0.06M. When eluting the target compound, the total amount of acidic agent 1 and acidic agent 2 is about 1% or less, preferably about 0.8% or less, more preferably about 0%, based on the total volume of eluent A and eluent B. 28. A method according to claim 26 or 27, wherein the amount is less than or equal to .6%, such as less than or equal to about 0.4%, in particular less than or equal to about 0.285%. The chromatography in step (4) is reverse phase chromatography, and/or the stationary phase of the reverse phase chromatography is selected from alkyl-bonded silica gel species, and/or the mobile phase in step (4) is eluent C; is eluent D,
Eluent C is water optionally containing acidic agent 3;
Eluent D is an organic solvent 2 optionally containing an acidic agent 4;
For the separation range, D is a slope from about 0% to 30% to about 30% to 100%, and the rest is C;
29. A method according to any one of claims 21 to 28, wherein the acidic agent 3 and acidic agent 4 are independently inorganic or organic acids. Acidic agent 3 is selected from TFA, phosphate buffer, ammonium acetate (AA), AcOH, H ₃ PO ₄ , TEAP and L-tartaric acid (L-TA), preferably TFA, phosphate buffer, selected from AA and TEAP, more preferably selected from TFA, phosphate buffer and TEAP, especially TFA, and/or the amount of acidic agent 3 is about 0.05% based on the total volume of eluent C. 01% to about 1%, preferably about 0.05% to about 0.5%, more preferably about 0.05% to about 0.3%, especially 0.1%. 29. The method according to any one of 29. acidic agent 4 is not present, or the type of acidic agent 4 is similar to acidic agent 3, and/or the amount of acidic agent 4 is from about 0.01% to about 1% based on the total volume of eluent D. %, preferably from about 0.05% to about 0.5%, more preferably from about 0.05% to about 0.3%, especially 0.1%, and/or in the elution composition. 31. The method of claim 30, wherein the total amount of acidic agent 3 and acidic agent 4 (if present) is about 0.01-0.25M. At any point in the chromatography process, the total amount of acidic agent 3 and acidic agent 4 is about 0.01% to about 1%, preferably about 0.05%, based on the total volume of eluent C and eluent D. 32. A method according to claim 30 or 31, wherein the amount is from about 0.5% to about 0.5%, more preferably from about 0.05% to about 0.3%, especially 0.1%. Each moiety 2 in the four compounds contained in mixture 1 is the same,
Part 2 has the structure of the following formula (V),

where Q is a group containing at least one chiral center,
L ¹ is absent or C _1-10 alkylene group, C _3-10 cycloalkylene group, C _6-10 arylene group, _4-10 membered heterocyclylene group, 5-10 membered heteroarylene group, -NH- , -(CO)-, -NH(CO)-, and -(CO)NH-,
Preferably, Q is _{a C5-50} hydrocarbyl group;
One or more "CH ₂ " structures in the hydrocarbyl group may be -O-, -S-, -NH-, -C(=O)-, -, provided that a stable structure is formed. S(=O)-, -S(=O) ₂ -, -NH(C=O)-, -C(=O)NH-, -NH-, -S(=O)-, -S(= O) NH-, -NHS(=O) ₂ -, or -S(=O) ₂ NH-, optionally replaced by
one or more "CH" structures in said hydrocarbyl group are optionally replaced by N or P, provided that a stable structure is formed;
one or more carbon, sulfur, nitrogen or phosphorus atoms in Q are independently optionally substituted by oxo (=O);
Q is optionally substituted by at least one substituent selected from R ^q , each independently R ^a1 , ^{-OR a1} , -SR ^a1 , -NR ^a1 R ^b1 , -C(=O )OR ^a1 , -C(=O)NR ^a1 R ^b1 , -C(=O)R ^a1 , -S(=O) ₂ OR ^a1 , -S(=O) ₂ R ^a1 , -S(=O) ₂ NR ^a1 R ^b1 , -S(=O)R ^a1 , -C(=S)OR ^a1 , -C(=S)NR ^a1 R ^b1 , -C(=S)R ^a1 , -P(=O) (OR ^a1 )OR ^b1 , -C(=NR ^a1 )NR ^b1 R ^c1 ,
33. A method according to any one of claims 21 to 32, wherein R ^a1 , R ^b1 and R ^c1 are each independently selected from hydrogen and a C _1-6 alkyl group. Part 2 has the structure of the following formula (V-1),

wherein the payload is selected from the group consisting of hydrogen and a small molecule compound containing at least one chiral center;
Preferably, said small molecule compounds include enzyme inhibitors, enzyme activators, receptor modulators, toxins, glycans, PEG moieties, radionuclides, nucleic acids and analogs, tracer molecules, low molecular weight peptides, low molecular weight peptidomimetics. 33. The method of claim 32, wherein the method is selected from , low molecular weight antibodies and antibody fragments. Each moiety 3 in the four compounds contained in mixture 1 is the same, and/or moiety 3 has the structure of formula (VI) below,

In the formula, L ² is a bond, or a C _1-10 alkylene group, a C _{3-10 cycloalkylene group, a C 6-10} arylene group, a _4- to ₁₀ -membered heterocyclylene group, a 5- to 10-membered heteroarylene group , -NH-, -(CO)-, -NH(CO)-, and -(CO)NH-,
M includes (1) an amino acid sequence 1 containing 1 to 20 amino acids, and (2) a "-(C ₂ H ₄ -O) _i -" structure, where i is an integer of 1 to 100; (3) a divalent optionally present group Y selected from cleavable sequence 1, spacer Sp1 and combinations thereof;
the cleavable sequence comprises an enzyme-cleavable amino acid sequence 2, the cleavable sequence comprises 1 to 10 amino acids,
22. The method of claim 21, wherein Sp1 is selected from the group consisting of a spacer sequence comprising 1 to 20 amino acids, PAB, and combinations thereof. L ¹ does not exist or

and/or ^L2 is a combination;

36. A method according to any one of claims 34 to 35, selected from: M is selected from the group consisting of a spacer sequence comprising 1 to 20 amino acids, PAB, lysine, oligomeric glycine, oligomeric alanine, oligomeric glycine/alanine mixtures with a degree of polymerization of 3 to 10, and combinations thereof;
Preferably,
M is L(G) _n , where L is leucine (Leu), or
M is E(G) _n , where E is glutamic acid (Glu), or
M is G _n ;
G is glycine (Gly),
37. A method according to claim 35 or 36, wherein n is an integer from 3 to 10, in particular 3. M has an ionizable amino group, acidic agent 1 is AcOH,
The amount of acidic agent 1 is about 0.01% to about 1%, preferably about 0.05% to about 0.5%, more preferably about 0.1% to about 0.5%, most preferably about 0.1% to about 0.3%, especially 0.3%;
38. The method of claim 37, wherein acidic agent 2 is absent or acidic agent 2 is present in an amount such that a target gradient of pH value of the mobile phase is obtained. M has an ionizable amino group, acidic agent 3 is TFA,
The amount of acidic agent 3 is about 0.01% to about 1%, preferably about 0.05% to about 0.5%, more preferably about 0.05% to about 0.3%, especially 0.1%,
Acidic agent 4 is absent or acidic agent 4 is about 0.01% to about 1%, preferably about 0.05% to about 0.5%, more preferably about 0.05% to about 0.5%, based on the total volume of eluent D. present in an amount of about 0.05% to about 0.3%, in particular 0.1%, or the acidic agent 4 is present in an amount such that a target gradient of the pH value of the mobile phase is obtained. 39. The method according to any one of paragraphs 35 to 38. Step (1) comprises a ring-opening reaction of the thiosuccinimide group resulting in the ring-opened thiosuccinimide structure of moiety 1;
40. A method according to any one of claims 21 to 39, wherein mixture 1 is preferably the reaction mixture obtained in the ring-opening reaction. 41. The method according to any one of claims 21 to 40, wherein the four compounds contained in mixture 1 are isomeric compounds (i) to (iv), respectively.

Compounds (i) and (ii) are obtained as separate products in step (2),
22. Process according to claim 21, wherein compounds (iii) and (iv) are obtained as separate products in step (4). 22. Process according to claim 21, wherein in step (2) compounds (i) and (ii) are obtained as separate products and compounds (iii) and (iv) are obtained as mixture 2. 22. The method according to claim 21, wherein mixture 1 is a reaction mixture obtained by ring-opening reaction of thiosuccinimide groups in the following compounds.

A composition comprising a mixture of a compound of formula (XIII) and a compound of formula (XIV), for example a racemic mixture,

During the ceremony,
The payload is a cytotoxin moiety containing one or more chiral centers;
L ¹ is absent or C _1-10 alkylene group, C _3-10 cycloalkylene group, C _6-10 arylene group, _4-10 membered heterocyclylene group, 5-10 membered heteroarylene group, -NH- , -(CO)-, -NH(CO)-, and -(CO)NH-,
L ² is a bond, or C _1-10 alkylene group, C _3-10 cycloalkylene group, C _6-10 arylene group, _4-10 membered heterocyclylene group, 5-10 membered heteroarylene group, -NH one or more divalent groups selected from the group consisting of -, -(CO)-, -NH(CO)-, and -(CO)NH-,
M contains a ligase recognition motif;
The composition is substantially free (eg, at least 90%, at least 95%, at least 98% or at least 99% free) of a compound of Formula XI or a compound of XII.

The ligase recognition motif is
(a) LPXTG, for example, where X is any amino acid residue, such as LPETG;
46. The composition of claim 45, wherein G is selected from (b) G _n , wherein G is glycine (Gly) and n is an integer from 3 to 10. 47. A composition according to claim 45 or 46, wherein M is H-Gly-Gly-Gly-Lys-NH ₂ and M is linked to L ² via the ε-amino group on the lysine. L ¹ does not exist,
L ² is

48. A composition according to any one of claims 45 to 47, selected from: L ¹ does not exist,
L ² is

and
M is

and
During the ceremony,
Y is a bond or a spacer sequence of 1 to 20 amino acids;
n is an integer from 3 to 10, for example 3;
x is selected from the group consisting of hydrogen, -OH, _-NH2 , amino acid fragments containing 1 to 10 amino acids, and nucleotide fragments containing 1 to 10 nucleotides, such as -OH, _-NH2 , and 49. Any one of claims 45 to 48 selected from the group consisting of amino acid fragments comprising 1 to 10 amino acids, such as selected from the group consisting of -OH, _-NH2 and Gly, such as _-NH2 . The composition described in . 50. The composition of any one of claims 45-49, wherein the cytotoxin comprises a thiol group moiety capable of reacting with a maleimide moiety. the cytotoxin is a maytansinoid;
For example, DM1

51. The composition according to any one of claims 45 to 50. 48. A mixture of a compound of formula (iii) and a compound of formula (iv) substantially free of a compound of formula (i) and a compound of formula (ii), according to any one of claims 45 to 47. Composition of.

An antibody drug conjugate composition comprising a mixture, e.g. a racemic mixture, of an antibody drug conjugate comprising an open ring thiosuccinimide structure of formula (III) and an antibody drug conjugate comprising an open ring thiosuccinimide structure of formula (IV). There it is,

The composition is substantially free (e.g., at least 90%, at least 95%, at least 98% or at least 99% free) of a compound of formula (I) or a compound of formula (II). Gate composition.

54. The antibody-drug conjugate composition of claim 53, formed by reacting the composition of any one of claims 45-52 with an antibody in the presence of a ligase. A mixture of a compound having the formula (XIX-1-1) and a compound having the formula (XX-1-1),

During the ceremony,
The payload is as defined above, e.g. a cytotoxin moiety containing one or more chiral centers, e.g. the remainder of the maytansinoid molecule excluding the thiol moiety, e.g. the remainder of DM1 without the thiol moiety. is the part of
A is an antibody or antigen-binding fragment thereof optionally modified to have a ligase donor substrate recognition motif or a ligase acceptor substrate recognition motif, e.g., A is a human epidermal growth factor receptor 2 (HER2) an antibody that binds, for example trastuzumab, which is modified to have a ligase donor substrate recognition motif or a ligase acceptor substrate recognition motif;
55. The antibody drug conjugate composition of claim 52, 53 or 54, wherein z is an integer of 1 or 2. A is trastuzumab with a C-terminally modified light chain (LCCT _L ) modified by the introduction of a short peptide spacer such as -GA- and a ligase donor substrate recognition motif LPXTGJ, where J in LPXTGJ is absent or , or an amino acid fragment comprising 1 to 10 amino acids. The payload is DM1,
L ³ does not exist,
57. The antibody-drug conjugate composition according to claim 55 or 56, wherein formulas (XIX-1-1) to (XX-1-1) have structures (vii) and (viii), respectively.

58. A method of treating cancer in a patient in need of cancer treatment, the method comprising administering to the patient an effective amount of an antibody drug conjugate composition according to any one of claims 53 to 57. Said method. 59. The method of claim 58, wherein the cancer is a HER2-positive cancer, such as a HER2-positive breast cancer. 60. The method of claim 58 or 59, wherein the antibody drug conjugate composition is as claimed in claim 57.

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